Resilin material footwear and fabrication methods

ABSTRACT

A method for making an article of footwear includes placing a purified recombinant resilin composition in a mold with a cross-linking solution, incubating the recombinant resilin composition in the cross-linking solution to generate a solid resilin material, fabricating a cushioning element of the article of footwear including at least a portion of the solid resilin material, and assembling the cushioning element insole with at least an upper of the article of footwear.

CROSS-REFERENCE TO RELATED APPLICATION

The present application is a Division of U.S. patent application Ser.No. 16/513,135, filed on Jul. 16, 2019, and entitled “RESILIN MATERIALFOOTWEAR AND FABRICATION METHODS,” which claims the benefit of U.S.Provisional Application No. 62/700,197, entitled “Cross-LinkedElastomeric Proteins in Polar Nonaqueous Solvents and Uses Thereof,”filed on Jul. 18, 2018, the contents of which are incorporated byreference in their entireties.

This application is related to International Application No.PCT/US2018/013839, filed Jan. 16, 2018, which claims benefit of U.S.Provisional Application No. 62/446,230, filed on Jan. 13, 2017, thecontents of which are each incorporated in their entirety.

BACKGROUND

The present disclosure generally relates to footwear fabricated, atleast in part, using a solid resilin material comprising a cross-linkedrecombinant resilin and a polar nonaqueous solvent.

Due to its potential characteristics with respect to elastic efficiency,compressive elastic modulus, tensile elastic modulus, shear modulus,hardness, rebound, and compression set, resilin is of increasinginterest in generating materials. Resilins have many unique propertiescompared to petroleum-based elastomers. In particular, resilin is aprotein, and therefore can be biodegraded, which makes it moreenvironmentally friendly than petroleum-based polymers. Also, resilin isbiocompatible and can therefore be used in applications that involvecontact with humans or animals. Lastly, the mechanical properties ofrecombinant resilins can be tuned through varying protein sequence,protein structure, amount of intermolecular cross-linking and processingvariables to produce elastomers designed for a universe of specificapplications.

The usability of specifically processed solid resilin material asalternative to petroleum-based elastomers makes it particularly suitablefor use in goods and articles typically made from or incorporating suchelastomers. In one application footwear, including various types ofsneakers, incorporate different elastomers in various ways. Resilincompositions and methods of making the same that have desirablemechanical properties and are suitable for large-scale, efficientproduction are disclosed in co-pending, commonly-assigned U.S.Provisional Pat. No. 62/700,197, the entire disclosure of which isincorporated by reference herein. What is needed are solid resilinmaterials suited for the various portions of footwear that have been orcan be made from elastomer, as well as configurations for such portionsof footwear and methods for their fabrication that utilize the uniqueproperties of resilin.

SUMMARY

In at least one aspect of the disclosure, an article of footwearincludes an upper and a midsole coupled with the upper. The midsoleincludes at least a portion of a solid resilin material comprising across-linked recombinant resilin and a polar nonaqueous solvent.

In various embodiments, the solid resilin material may be an elastomerthat defines at least one physical property resembling that of apetroleum-based elastomer. In one example, the petroleum-based elastomermay be ethyl vinyl acetate foam.

In an additional or alternative embodiment, the midsole may define atleast one exposed ground-contacting surface. The at least one exposedground contacting surface may be in one of the heel or fore-foot areasof the midsole and may, further be uncovered by an outsole.

In various embodiments the article of footwear can be a sneaker, furtherincluding a lasting board, the upper being affixed with the lastingboard to define an interior foot-receiving cavity therewith, and themidsole being coupled with the upper opposite the lasting board. In afurther embodiment, the article of footwear can be a sandal, and theupper can include one or more straps and defines at least one open area.

In at least another aspect, a method for making an article of footwearincludes placing a purified recombinant resilin composition in a moldwith a cross-linking solution, incubating the recombinant resilincomposition in the cross-linking solution to generate a solid resilinmaterial, fabricating a midsole including at least a portion of thesolid resilin material, and assembling the midsole with an upper. Invarious embodiments, the method can further comprise, prior tofabricating the midsole, subjecting the solid resilin material to asolvent exchange process to substantially remove the cross-linkingsolution and configure the solid resilin material as a solid resilinmaterial comprising a cross-linked recombinant resilin and a polarnonaqueous solvent.

In at least another aspect, a method for making an article of footwearincludes placing a purified recombinant resilin composition in a moldwith a cross-linking solution, incubating the recombinant resilincomposition in the cross-linking solution to generate a solid resilinmaterial, fabricating an insole including at least a portion of thesolid resilin material, and assembling the insole within an upper andover a portion of a midsole of the article of footwear.

In at least another aspect, a method for making an article of footwearincludes placing a purified recombinant resilin composition in a moldwith a cross-linking solution, incubating the recombinant resilincomposition in the cross-linking solution to generate a solid resilinmaterial, fabricating a cushioning element of the article of footwearincluding at least a portion of the solid resilin material, andassembling the cushioning element insole with at least an upper of thearticle of footwear.

In at least another aspect, an insole for an article of footwearincludes a solid resilin material comprising a cross-linked recombinantresilin and a polar nonaqueous solvent and defining at least a portionof the insole. In an embodiment, the portion of the insole comprisingthe solid resilin material can include an exposed foot-supportingsurface. Additionally or alternatively, the portion of the insolecomprising the solid resilin material can define an overall shape of theinsole.

These and other features, advantages, and objects of the present devicewill be further understood and appreciated by those skilled in the artupon studying the following specification, claims, and appendeddrawings.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing summary, as well as the following detailed description ofthe invention, will be better understood when read in conjunction withthe appended drawings. For the purpose of illustration, there are shownin the drawings, certain aspects of the disclosure. It should beunderstood, however, that the disclosure is not limited to the precisearrangements and instrumentalities shown. Drawings are not necessarilyto scale. Certain features of the invention may be exaggerated in scaleor shown in schematic form in the interest of clarity and conciseness.

In the drawings:

FIG. 1 is a front perspective view of a sneaker according to an aspectof the disclosure;

FIG. 2 is a front perspective exploded view of the sneaker;

FIG. 3 is a front perspective exploded view of an midsole of thesneaker;

FIG. 4 is a front perspective view of two samples of foamed resilinmaterial;

FIGS. 5A and 5B are exploded perspective and top elevation views of alaminated perforated structure of solid resilin material;

FIG. 6 is a top elevation view of a further example of a laminatedperforated structure of solid resilin material;

FIGS. 7A and 7B are exploded perspective and top elevation views of afurther example a laminated perforated structure of solid resilinmaterial;

FIG. 8 is a front perspective view of a sneaker according to anotheraspect of the disclosure;

FIG. 9 is a front perspective exploded view of the sneaker;

FIG. 10 is a bottom perspective view of the sneaker;

FIG. 11 is a front perspective view of a sandal according to an aspectof the disclosure;

FIG. 12 is a front perspective view of an alternative sandal accordingto an aspect of the disclosure;

FIG. 13 is a front perspective view of a sneaker according to anotheraspect of the disclosure;

FIG. 14 is a front perspective exploded view of the sneaker; and

FIG. 15 is a bottom perspective view of a portion the sneaker.

DETAILED DESCRIPTION OF EMBODIMENTS

The details of various embodiments of the invention are set forth in thedescription below. Other features, objects, and advantages of theinvention will be apparent from the description and the drawings, andfrom the claims.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of ordinary skillin the art to which this disclosure pertains.

The terms “a” and “an” and “the” and similar referents as used hereinrefer to both the singular and the plural, unless otherwise indicatedherein or clearly contradicted by context.

The term “about,” “approximately,” or “similar to” means within anacceptable error range for the particular value as determined by one ofordinary skill in the art, which can depend in part on how the value ismeasured or determined, or on the limitations of the measurement system.It should be understood that all ranges and quantities described beloware approximations and are not intended to limit the invention. Whereranges and numbers are used these can be approximate to includestatistical ranges or measurement errors or variation. In someembodiments, for instance, measurements could be plus or minus 10%.

Amino acids can be referred to by their single-letter codes or by theirthree-letter codes. The single-letter codes, amino acid names, andthree-letter codes are as follows: G—Glycine (Gly), P—Proline (Pro),A—Alanine (Ala), V—Valine (Val), L—Leucine (Leu), I—Isoleucine (Ile),M—Methionine (Met), C—Cysteine (Cys), F—Phenylalanine (Phe), Y—Tyrosine(Tyr), W—Tryptophan (Trp), H—Histidine (His), K—Lysine (Lys), R—Arginine(Arg), Q—Glutamine (Gin), N—Asparagine (Asn), E—Glutamic Acid (Glu),D—Aspartic Acid (Asp), S—Serine (Ser), T—Threonine (Thr).

The terms “including,” “includes,” “having,” “has,” “with,” or variantsthereof are intended to be inclusive in a manner similar to the term“comprising.”

The term “microbe” as used herein refers to a microorganism, and refersto a unicellular organism. As used herein, the term includes allbacteria, all archaea, unicellular protista, unicellular animals,unicellular plants, unicellular fungi, unicellular algae, all protozoa,and all chromista.

The term “native” as used herein refers to compositions found in naturein their natural, unmodified state.

The terms “optional” or “optionally” mean that the feature or structuremay or may not be present, or that an event or circumstance may or maynot occur, and that the description includes instances where aparticular feature or structure is present and instances where thefeature or structure is absent, or instances where the event orcircumstance occurs and instances where the event or circumstance doesnot occur.

The term “secreted fraction” as used herein refers to the fraction ofrecombinant resilins that are secreted from cells compared to the totalresilins produced by the cells.

The term “secretion signal” as used herein refers to a short peptidethat when fused to a polypeptide mediates the secretion of thatpolypeptide from a cell.

The term “secreted resilin coding sequence” as used herein refers to anucleotide sequence that encodes a resilin as provided herein fused atits N-terminus to a secretion signal and optionally at its C-terminus toa tag peptide or polypeptide.

The term “recombinant” as used herein in reference to a polypeptide(e.g., resilin) refers to a polypeptide that is produced in arecombinant host cell, or to a polypeptide that is synthesized from arecombinant nucleic acid.

The term “recombinant host cell” as used herein refers to a host cellthat comprises a recombinant nucleic acid.

The term “recombinant nucleic acid” as used herein refers to a nucleicacid that is removed from its naturally occurring environment, or anucleic acid that is not associated with all or a portion of a nucleicacid abutting or proximal to the nucleic acid when it is found innature, or a nucleic acid that is operatively linked to a nucleic acidthat it is not linked to in nature, or a nucleic acid that does notoccur in nature, or a nucleic acid that contains a modification that isnot found in that nucleic acid in nature (e.g., insertion, deletion, orpoint mutation introduced artificially, e.g., by human intervention), ora nucleic acid that is integrated into a chromosome at a heterologoussite. The term includes cloned DNA isolates and nucleic acids thatcomprise chemically-synthesized nucleotide analog.

The term “vector” as used herein refers to a nucleic acid moleculecapable of transporting another nucleic acid to which it has beenlinked. One type of vector is a “plasmid,” which generally refers to acircular double stranded DNA loop into which additional DNA segments canbe ligated, but also includes linear double-stranded molecules such asthose resulting from amplification by the polymerase chain reaction(PCR) or from treatment of a circular plasmid with a restriction enzyme.Other vectors include bacteriophages, cosmids, bacterial artificialchromosomes (BAC), and yeast artificial chromosomes (YAC). Another typeof vector is a viral vector, wherein additional DNA segments can beligated into the viral genome. Certain vectors are capable of autonomousreplication in a cell into which they are introduced (e.g., vectorshaving an origin of replication that functions in the cell). Othervectors can be integrated into the genome of a cell upon introductioninto the cell, and are thereby replicated along with the cell genome.

The term “repeat” as used herein, in reference to an amino acid ornucleic acid sequence, refers to a sub-sequence that is present morethan once in a polynucleotide or polypeptide (e.g., a concatenatedsequence). A polynucleotide or polypeptide can have a direct repetitionof the repeat sequence without any intervening sequence, or can havenon-consecutive repetition of the repeat sequence with interveningsequences. The term “quasi-repeat” as used herein, in reference to aminoacid or nucleic acid sequences, is a sub-sequence that is inexactlyrepeated (i.e., wherein some portion of the quasi-repeat subsequence isvariable between quasi-repeats) across a polynucleotide or polypeptide.Repeating polypeptides and DNA molecules (or portions of polypeptides orDNA molecules) can be made up of either repeat sub-sequences (i.e.,exact repeats) or quasi-repeat sub-sequences (i.e., inexact repeats).

The term “native resilin” as used herein refers to an elastomericpolypeptide or protein produced by insects. GenBank Accession Nos. ofnon-limiting examples of native resilin includes the following NCBIsequence numbers: XP 002034179 (Drosophila sechellia), NP 995860(Drosophila melanogaster), NP 611157 (Drosophila melanogaster), Q9V7U0(Drosophila melanogaster), AAS64829, AAF57953 (Drosophila melanogaster),EGI57805, AEQ49438, XP003399675, AEQ49434, AEQ49437, XP 012058333, XP006563165, XP 011184157, XP 001843145, XP 015011737, XP 008209097, XP001605137, XP 002428637, XP 011165933, NP 001182329, XP 014220291, andADM26717.

The term “modified” as used herein refers to a protein or polypeptidesequence that differs in composition from a native protein orpolypeptide sequence, where the functional properties are preserved towithin 10% of the native protein or polypeptide properties. In someembodiments, the difference between the modified protein or polypeptideand the native protein or polypeptide can be in primary sequence (e.g.,one or more amino acids are removed, inserted or substituted) orpost-translation modifications (e.g., glycosylation, phosphorylation).Amino acid deletion refers to removal of one or more amino acids from aprotein. Amino acid insertion refers to one or more amino acid residuesbeing introduced in a protein or polypeptide. Amino acid insertions maycomprise N-terminal and/or C-terminal fusions as well as intra-sequenceinsertions of single or multiple amino acids. Amino acid substitutionincludes non-conservative or conservative substitution, whereconservative amino acid substitution tables are well known in the art(see for example Creighton (1984) Proteins. W. H. Freeman and Company(Eds)). In some embodiments, the modified protein or polypeptide and thenative protein or polypeptide amino acid or nucleotide sequence identityis at least 60%, at least 65%, at least 70%, at least 75%, at least 80%,at least 85%, at least 90%, at least 95%, or at least 98% of the aminoacids or nucleotide bases.

The term “truncated” as used herein refers to a protein or polypeptidesequence that is shorter in length than a native protein or polypeptide.In some embodiments, the truncated protein or polypeptide can be greaterthan 10%, or greater than 20%, or greater than 30%, or greater than 40%,or greater than 50%, or greater than 60%, or greater than 70%, orgreater than 80%, or greater than 90% of the length of the nativeprotein or polypeptide.

The term “homolog” or “substantial similarity,” as used herein, whenreferring to a polypeptide, nucleic acid or fragment thereof, indicatesthat, when optimally aligned with appropriate amino acid or nucleotideinsertions or deletions with another amino acid or nucleic acid (or itscomplementary strand), there is amino acid or nucleotide sequenceidentity in at least 60%, at least 65%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, or at least 98% ofthe amino acids or nucleotide bases, as measured by any well-knownalgorithm of sequence identity, such as FASTA, BLAST or Gap, asdiscussed above.

The term “resilin” as used herein refers to a protein or a polypeptide,capable of cross-linking to form an elastomer, where the protein orpolypeptide is a native resilin, or a native resilin that is modified,or a native resilin that is truncated. Resilins of the present inventionare preferably recombinant resilins. In some embodiments, recombinantresilins comprise a natural or modified (e.g., truncated orconcatenated) nucleotide sequence coding for resilin or resilinfragments (e.g., isolated from insects), heterologously expressed andsecreted from a host cell. In preferred embodiments, the secretedrecombinant resilin protein is collected from a solution extracellularto the host cell.

As used herein, the term “elastomer” refers to a polymer withviscoelasticity and typically weak inter-molecular forces (except forcovalent cross-links between molecules, if they are present).Viscoelasticity is a property of materials that exhibit both viscous andelastic characteristics when undergoing deformation, and thereforeexhibit time-dependent strain. Elasticity is associated with bondstretching along crystallographic planes in an ordered solid, andviscosity is the result of the diffusion of atoms or molecules inside anamorphous material. Elastomers that are viscoelastic, therefore,generally have low Young's modulus and high failure strain compared withother materials. Due to the viscous component of the material,viscoelastic materials dissipate energy when a load is applied and thenremoved. This phenomenon is observed as hysteresis in the stress-straincurve of viscoelastic materials. As a load is applied there is aparticular stress-strain curve, and as the load is removed thestress-strain curve upon unloading is different than that of the curveduring loading. The energy dissipated is the area between the loadingand unloading curves.

As used herein, the term “nonaqueous” refers to a solvent thatpredominantly comprises one or more compounds that are not water. Thisincludes compositions that have undergone a solvent exchange processwith a solvent that results in an overall decrease in the proportion ofwater present as a solvent, i.e., water has been replaced by non-watermolecules as a solvent. In some embodiments, a nonaqueous solvent is onethat comprises less than 50% water. A polar nonaqueous solvent, as usedherein with respect to solvents for cross-linked resilin compositions,refers to any nonaqueous solvent that is capable of dissolving resilin.

As used herein, the term “coupled” (in all of its forms, couple,coupling, coupled, etc.) generally means the joining of two componentsdirectly or indirectly to one another. Such joining may be stationary innature or movable in nature. Such joining may be achieved with the twocomponents and any additional intermediate members being integrallyformed as a single unitary body with one another or with the twocomponents (e.g., the upper may be coupled to the outsole directly orthrough the midsole positioned therebetween). Such joining may bepermanent in nature or may be removable or releasable in nature unlessotherwise stated.

Recitation of ranges of values herein are merely intended to serve as ashorthand method of referring individually to each separate valueinclusively falling within the range, unless otherwise indicated herein,and each separate value is incorporated into the specification as if itwere individually recited herein.

When referring to the drawings, it is to be understood that the depictedarticle may assume various alternative orientations, except whereexpressly specified to the contrary. It is also to be understood thatthe specific articles, components, and processes illustrated in theattached drawings, and described in the following specification aresimply exemplary of the concepts defined in the appended claims. Hence,specific dimensions and other physical characteristics relating to theembodiments disclosed herein are not to be considered as limiting,unless the claims expressly state otherwise. The details of variousembodiments are set forth in the description below. Other features,objects, and advantages will be apparent from the description. Unlessotherwise defined herein, scientific and technical terms used shall havethe meanings that are commonly understood by those of ordinary skill inthe art. Further, unless otherwise required by context, singular termsshall include the plural and plural terms shall include the singular.The terms “a” and “an” includes plural references unless the contextdictates otherwise. Generally, nomenclatures used in connection with,and techniques of, biochemistry, enzymology, molecular and cellularbiology, microbiology, genetics and protein and nucleic acid chemistryand hybridization described herein are those well-known and commonlyused in the art.

Exemplary methods and materials are described below, although methodsand materials similar or equivalent to those described herein can alsobe used and will be apparent to those of skill in the art. Allpublications and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentspecification, including definitions, will control. The materials,methods, and examples are illustrative only and not intended to belimiting.

Referring to the embodiment illustrated in FIG. 1, reference numeral 10generally designates an article of footwear, specifically in the form ofa sneaker. The sneaker 10 includes an upper 12 and a midsole 14 affixedwith the upper 12. The midsole 14 includes at least a portion of aresilin material comprising a cross-linked recombinant resilin solid anda polar nonaqueous solvent.

Recombinant Resilin Materials and Production Methods

Provided herein is an overview of general compositions comprisingrecombinant resilins, and methods for their production that, unlessotherwise indicated, may be common among various end-products that mayuse recombinant resilins, including those described below. Thesecompositions and methods are generally similar to those described in theabove-referenced '197 Application. In this respect, examples and detailsregarding the various aspects of the compositions and methods furtheringthe present disclosure are described therein.

Resilins have many unique properties compared to petroleum-basedelastomers. Most notably, at least in its many naturally-occurringapplications, resilin has an extreme elastic efficiency (i.e.,resilience), where very little of the energy input into deformation islost as heat. Other desirable properties of resilin relate to, forexample, resilin's compressive elastic modulus, tensile elastic modulus,shear modulus, hardness, rebound, and compression set. Moreover, resilinis a protein, and therefore can be biodegraded, which makes it moreenvironmentally friendly than petroleum-based polymers. Also, resilin isbiocompatible and can therefore be used in applications that involvecontact with humans or animals. Lastly, the mechanical properties ofrecombinant resilins can be tuned through varying protein sequence,protein structure, amount of intermolecular cross-linking and processingvariables to produce elastomers designed for a universe of specificapplications.

Described herein are various cross-linked resilin compositions withvarious mechanical properties and methods of producing them. Alsoprovided herein are methods of cross-linking resilin compositions toform various examples of a cross-linked resilin solid that can beperformed in large batches and results in little to no degradation fromimpurities left over from the cross-linking reaction in comparison toprevious methods. In some examples, the cross-linking reaction comprisesexposure of the resilin to a persulfate, such as ammonium persulfate.Heat can be applied to initiate a cross-linking reaction catalyzed bypersulfate. In some examples, cross-linking occurs in vessels or moldssuch that the recombinant resilin compositions obtained have specificshapes or forms, as discussed in the various practical examplesdiscussed below and shown in the figures.

The cross-linked resilin solid compositions provided herein also includecross-linked resilin compositions comprising a polar nonaqueous solventto provide selected mechanical properties with respect to elasticmodulus, hardness, maximum elastic compressive load, resilience,material lifetime/fatigue, or the like, that are determined to besuitable for use in certain applications, including in the examples offootwear, as discussed below. In some embodiments, the compositions aremade by performing a solvent exchange with a resilin composition toreplace an aqueous solvent with a nonaqueous solvent. In otherapplications, a solvent exchange may be made to replace a cross-linkingsolution or solvent with a solvent that is selected for the propertiesit contributes to a finished product, including resistance todegradation, that may not be realized by the cross-linking solution.Solvents that are capable of solvent exchange with cross-linked resilininclude solvents that dissolve resilin in its non-cross-linked form.

In some examples, the nonaqueous solvent is non-volatile and watersoluble or polar. In some embodiments, the molecular weight of thesolvent is about 100 or less. In further examples, the polar nonaqueoussolvent comprises non-volatile water miscible solvents mixed with wateror used as neat solutions such as propylene glycol, glycerol, ethyleneglycol, polyethylene glycol of various molecular weights from 400 to 1million. In another example, the polar nonaqueous solvent may compriseB) Ionic liquids as neat solutions or mixed with water (in ratios from70:30 IL:water to 30:70 IL:water) such as 1-ethyl-3-methylimidazoliumacetate and 1-butyl-3-methylimidazolium bromide. Notably,1-ethyl-3-methylimidazolium acetate not only dissolves resilin at 20 wt% when mixed 1:1 with water, but the crosslinking reaction can also becarried out in this solvent when mixed with water. This solution doesnot dehydrate over time due to the ionic liquid's hygroscopic nature.Examples of resilin materials with other polar nonaqueous solvents arepossible, additional examples of which are given in the '197Application.

Resilin Compositions

Examples of native resilin may contain an N-terminal A-domain comprisinga plurality of repeat units comprising the consensus amino acid sequenceYGXP (“A-repeat”), where X is any amino acid; a chitin-binding type RR-2(C) domain; and a C-terminal B-domain comprising a plurality of repeatunits comprising the consensus amino acid sequence UYZXZ (“B-repeat”),where U is glycine or serine; Z is serine, glycine, arginine, orproline; and X is any amino acid. Not all naturally occurring resilinshave A-, C-, and B-domains. Native resilins produced by various insectstypically have inexact repeats (i.e., quasi-repeats) within the A-and/or B-domains with some amino acid variation between thequasi-repeats. Various examples of recombinant resilins according to thepresent disclosure can similarly comprise one or more A-repeats and oneor more B-repeats in various consensus sequences of motifs of aminoacids and residues described in greater detail in the above-incorporated'197 application. Additionally, some examples of the recombinantresilins comprise one or more A-repeats, one or more B-repeats, and/orone or more C-domain. In further examples, the recombinant resilinscomprise: one or more A-repeats or one or more B-repeats but not both;one or more A-repeats but not B-repeats or C-domains; one or moreB-repeats but not A-repeats or C-domains. In examples in which therecombinant resilins comprise a C-domain, the C-domain can be situatedeither on the N-terminal or the C-terminal sides of the A-repeats orB-repeats, or between the A-repeats and the B-repeats. Some examples ofthe recombinant resilins may further comprise additional the sequencescontaining an amino acid, which may be located on the N-terminal side ofan A-repeat or B-repeat.

In some examples, the recombinant resilins are full-length nativeresilins expressed in a non-native environment. In some embodiments, therecombinant resilins comprise a truncated version of native resilins. Insome embodiments, the truncated native resilins comprise at least oneA-repeat. In some embodiments, the truncated native resilins comprise atleast one B-repeat. Non-limiting examples of full-length and truncatednative resilins are provided in the above-referenced '197 Application.In some examples, the recombinant resilins are full-length or truncatednative resilins that are cross-linked in a non-native manner (e.g., lessor more cross-linking, cross-linking via different amino acid residues).In some of the examples, the recombinant resilins are modifiedfull-length or native resilins that are truncated to various degrees.

In some examples, the modified resilins differ from full-length ortruncated native resilins in amino acid residues that arepost-translationally modified (e.g., glycosylated, phosphorylated) suchthat the modified resilins have one or more different locations and/ordifferent amounts and/or different types of post-translationalmodifications than the full-length or truncated native resilins. In someembodiments, the modified resilins differ from full-length or truncatednative resilins in amino acid residues that are involved incross-linking such that the modified resilins have one or more differentlocations and/or different amounts and/or different types of amino acidsthat are involved in cross-linking than full-length or truncated nativeresilins. In some such embodiments, the modified resilins differ fromthe full-length or truncated native resilin in comprising one or moreadditional or fewer tyrosine residues, one or more additional or fewerlysine residues, and/or one or more additional or fewer cysteineresidues.

In some examples, the recombinant resilins comprise concatenated nativeor truncated native resilins or concatenated modified resilins. In someexamples, the concatenated native or truncated native resilins orconcatenated modified resilins comprise at least 2 A-repeats (e.g., 2,3, 4, 5, 6, 7, 8, 9, 10, or more). In some embodiments, the concatenatedtruncated native resilins or concatenated modified resilins comprise atleast 2 B-repeats (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more).

Cross-Linking

In some embodiments, the recombinant resilins can be cross-linkedaccording to various methods to obtain specific recombinant resilincompositions. The recombinant resilin in the desired solvent withcross-linking agents can be filled into molds to achieve a desired shapeof the resulting solid after cross-linking. Examples of resultingrecombinant resilin solids are in FIGS. 1-15 and are discussed furtherbelow.

In various examples, cross-linking may be achieved via tyrosine residuesto create di- and tri-tyrosine crosslinking in resilin to form a resilinsolid. In other examples, cross linking can be achieved via lysineresidues. In some examples, cross linking can be achieved via cysteineresidues. In some examples, cross-linking may employ transglutaminase orpoly(ethylene glycol) (PEG). In other examples, recombinant resilin canbe cross-linked via enzymatic cross-linking (e.g., using horseradishperoxidase). While this method can efficiently cross-link largesolutions of resilin, the resulting cross-linked product comprisescovalently incorporated active enzyme in the cross-linked resilin solid.This may yields radical chain reactions that could cause degradation ofthe protein backbone of the resilin, if left in a resulting resilinsolid. In other examples, recombinant resilin can be cross-linked viaphotochemical cross-linking, although such cross-linking may not beefficient for reactions where photoactivation throughout the mold isrequired.

Examples of additional cross-linking chemistries are disclosed in the'197 Application that may prevent degradation and make solid substanceswith some mechanical properties preferred for certain applications wherethe amount and form of energy absorption is important. In some suchexamples, recombinant resilin may be cross-linked via a solventcomprising ammonium persulfate (at various concentration) andapplication of heat (e.g., incubation at a temperature of about 80° C.for about 2.5 hours, with other examples of heats and incubationtemperatures provided therein). In some examples, other persulfates maybe used.

Solvent-Exchanged Resilin Solids

Cross-linked resilin can be formed in an aqueous solvent resulting in acomposition that has a low hardness and elastic modulus that is lesssuitable for certain applications where energy absorption and stiffnessare desired. In some examples, a solvent exchange may be performed oncross-linked resilin compositions to replace an aqueous solvent with apolar nonaqueous solvent to provide desired material properties. Asdiscussed above, the polar nonaqueous solvent may comprise non-volatilewater miscible solvents mixed with water or used as neat solutions suchas propylene glycol, glycerol, ethylene glycol, polyethylene glycol ofvarious molecular weights from 400 to 1 million. In another example, thepolar nonaqueous solvent may comprise ionic liquids as neat solutions ormixed with water (in ratios from 70:30 IL:water to 30:70 IL:water) suchas 1-ethyl-3-methylimidazolium acetate and 1-butyl-3-methylimidazoliumbromide. As also discussed above, 1-ethyl-3-methylimidazolium acetatenot only dissolves resilin at 20 wt % when mixed 1:1 with water, but thecrosslinking reaction can also be carried out in this solvent when mixedwith water. This solution does not dehydrate over time due to the ionicliquid's hygroscopic nature and low vapor pressure. As described herein,material properties of cross-linked resilin compositions, includingelastic modulus, hardness, maximum elastic compressive load, resilience,and material lifetime/fatigue, can be tuned using solvent exchange.Solvents that are capable of doing solvent exchange with cross-linkedresilin include solvents that dissolve resilin in its non-crosslinkedform. Additionally, as discussed further below, the exchange of a lowvapor pressure solvent for water also increases the duration of time forwhich the resilin material remains elastomeric, as resilin relies on acertain level of hydration to remain elastomeric. An aqueous solventwill evaporate over a relatively short duration of time, such that theresilin becomes a hard solid. Polar nonaqueous solvents are moreresistant to evaporation, limiting the effects thereof on the resultingresilin material.

In some examples, a solvent exchange to replace an aqueous solvent ofresilin with a polar nonaqueous solvent can be performed in the presenceof heat, e.g., at a temperature of about 60° C. In some embodiments thesolvent exchange process is performed in a solution containing at least1×, at least 2×, at least 5×, at least 10×, or at least 20× the volumeof exchange solvent relative to the resilin solid. In some embodiments,the solvent exchange is performed for at least 1 hour, at least 2 hours,at least 4 hours, at least 8 hours, at least 16 hours, at least 24hours, or at least 48 hours. In some examples, glycerol, propyleneglycol, ethylene glycol, or DMSO are used as exchange solvents forcross-linked resilin solid compositions.

In some examples, the choice of exchange solvent and the concentrationused can be selected to achieve a desired tunable mechanical property,such as stiffness, abrasion resistance, and the like, from the solventdesign. This can be selected depending on the desired application (e.g.,shoe outers, golf balls, etc.). Further, examples of the cross-linkedresilin compositions formed by the above solvent exchange process and ina nonaqueous solvent may be stable at room temperature for extendedperiods of time compared to water-based cross-linked resilin solids.Further water-based resilin solids may exhibit lower material strengthcompared to nonaqueous solvent-based resilin solids. Still further, therecombinant resilin compositions discussed herein may have an elasticmodulus greater than cross-linked recombinant resilin in an aqueoussolvent.

In some implementations, the cross-linked resilin compositions describedherein can have a Shore 00 Hardness of 50 or more, 40 or more, 30 ormore, 20 or more, 10 or more, from 10 to 50, 40, 30, or 20; from 20 to50, 40, or 30; from 30 to 50 or 40; or from 40 to 50 (which can bemeasured according to ASTM D2240). In further examples, the recombinantresilin compositions may have a hardness of at least 10 (as measuredusing a Shore 00 Durometer via ASTM D2240) or a hardness of from about10 to about 50 (as measured using a Shore 00 Durometer via ASTM D2240).

In further examples, the recombinant resilin compositions describedherein may exhibit a rebound resilience from about 40% to about 60% (asmeasured by ASTM D7121). In further examples, the recombinant resilincompositions described herein may exhibit a compressive stress at 25% ofabout 6 psi to about 8 psi (as measured by ASTM D575). In furtherexamples, the recombinant resilin compositions described herein may notundergo an elastic to plastic transition below 2 kN of compressive force(as measured by a Zwick compression test).

The solid material properties of the various specific resilincompositions achieved according to the above, such as resilience,compressive elastic modulus, tensile elastic modulus, shear modulus,extension to break, maximum tensile strength, hardness, stiffness, andrebound may be be tuned based on the solvent used and how the solventexchange is performed. The concentration of resilin in the resilin solidand the amount of full length resilin as a portion of total resilin canalso be adjusted to affect the material properties of the cross-linkedresilin solid composition. In various examples, solvent exchange toreplace the water-based resilin composition (i.e, a cross-linked resilincomposition in an aqueous solvent) with a polar nonaqueous-based resilincomposition (i.e., a cross-linked resilin composition in a polarnonaqueous solvent), as described herein, may result in a stiffermaterial with a similar resilience and a similar elasticity.

Foamed Resilin Solids

In some embodiments, the recombinant resilin composition is a foammaterial. In some embodiments, a method of preparing the recombinantresilin foam, comprises: providing a cross-linked recombinant resilinsolid composition in an aqueous solvent; exchanging said aqueous solventwith a polar nonaqueous solvent; and introducing one or more bubbles tothe cross-linked recombinant resilin solid composition. Any method ofintroducing bubbles known in the art may be used herein. For instance,methods of introducing bubbles include, but are not limited to,vortexing, mixing, adding yeast, and chemical reactions. In someembodiments, the introducing the one or more bubbles may occur at thesame time the cross-linked recombinant resilin solid composition isprovided. In some embodiments, the introducing the one or more bubblesmay occur after the cross-linked recombinant resilin solid compositionis provided.

Blowing agents typically are introduced into polymeric material to makepolymer foams in one of two ways. According to one technique, a chemicalblowing agent is mixed with a polymer. The chemical blowing agentundergoes a chemical reaction in the polymeric material, typically underconditions in which the polymer is molten, causing formation of a gas.Chemical blowing agents generally are low molecular weight organiccompounds that decompose at a particular temperature and release a gassuch as nitrogen, carbon dioxide, or carbon monoxide.

Exemplary chemical blowing agents include, but are not limited to,sodium bicarbonate, potassium bicarbonate, ammonium, azodicarbonamide,isocyanate, hydrazine, isopropanol, 5-phenyltetrazole, triazole,4,4′oxybis(benzenesulfonyl hydrazide) (OBSH), trihydrazine triazine(THT), hydrogen phosphate, tartaric acid, citric acid, andtoluenesulphonyl semicarbazide (TSS).

In some embodiments, foaming agents, thickeners, and/or hardeners areadded to the recombinant resilin solid. Exemplary foaming agentsinclude, but are not limited to, xanthan gum, sodium dodecyl sulfate,ammonium lauryl sulfate, bovine serum albumin. Exemplary thickenersinclude, but are not limited to, fumed silica and xanthan gum. Exemplaryhardeners include, but are not limited to, aliphatic polyamine, fattypolyamides, aromatic polyamine hardeners, anhydride hardeners, borontrifluoride hardeners, and curing agents (dicyandiamide).

According to another technique a physical blowing agent, i.e., a fluidthat is a gas under ambient conditions, is injected into a moltenpolymeric stream to form a mixture. The mixture is subjected to apressure drop, causing the blowing agent to expand and form bubbles(cells) in the polymer. In some embodiments, the pressure required isabout 500 psi to about 2000 psi, e.g., about 600 psi to about 1000 psi,about 700 psi to about 1500 psi, and about 800 psi to about 2000 psi. Insome embodiments, the pressure required is about 500 psi.

Exemplary physical blowing agents include, but are not limited to,chlorofluorocarbon (CFC), dissolved nitrogen, N2, CH4, H2, CO2, Ar,pentane, isopentane, hexane, methylene dichloride, anddichlorotetra-fluoroethane.

Mechanical Properties

Further examples of resilin compositions that may be derived by thepresent disclosure may have different properties compared tocompositions comprising cross-linked resilins. In some examples, thecompositions provided herein may have similar properties compared tosynthetic elastic or elastomeric materials, including various foams andthe like. Non-limited examples of such properties include resilience,compressive elastic modulus, tensile elastic modulus, shear modulus,hardness, rebound, and compression set. Parameters that can be modifiedto obtain compositions with specific mechanical properties include, forexample, the length and/or sequence of the recombinant resilins, theextent and/or type of post-translational modifications of therecombinant resilins, the extent and/or type of cross-linking of therecombinant resilins and the nature of the solvent of the cross-linkedresilin composition.

Mechanical properties such as maximum tensile strength, compressiveelastic modulus, tensile elastic modulus, shear modulus, extension tobreak and resilience can be measured using many different types oftensile and compression systems that conduct stress-strain measurementson elastomeric samples. Various possible processes and methods fortesting these properties and various values for the resilin compoundsthat can be derived according to the present disclosure are described inthe '197 Application.

The compositions provided herein have a number of uses, including butnot limited to applications in aerospace, automotive, sportingequipment, vibration isolation, footwear, and clothing among others.Some applications from these categories are listed as non-limitingexamples. Due to the desirable elastic efficiency, resilin can be usedas an energy storage device (e.g., a rubber band) for storing andrecovering mechanical energy. Automobile suspension systems can beimproved by application of resilin bushings to keep more tire contact onthe road when going over bumps and through potholes at speed.Additionally, there are a number of sporting equipment applications forresilin with differently tuned mechanical properties including cores ofgolf balls, tennis racket grips, golf club grips, and table tennispaddles.

An application of particular interest is footwear due to the uniqueproperties of resilin compositions provided herein. As an insole ormidsole, resilin can improve the comfort and bioefficiency of shoes bycushioning the foot strike and restoring more of the energy from thatfootstrike as forward momentum. As a midsole, resilin can make up theentire midsole or be encapsulated within another material to complementits properties (e.g., an abrasion or wear resistant material, or amaterial tuned for traction). The resilin midsole can also contain aplurality of resilin materials with differently tuned mechanicalproperties that work in concert to provide enhanced performance (e.g.,softer heel strike area and firmer arch support), as discussed furtherbelow.

Vectors, Host Cells, and Fermentations

As disclosed further in the '197 Application, recombinant host cellscomprising disclosed vectors may be used in fermentations to produceresilin protein, including those according to the various sequencesdiscussed above. In some examples, the vectors provided comprisesecreted resilin coding sequences, which encode a resilin polypeptidefused at its N-terminus to a secretion signal and optionally at itsC-terminus to a tag peptide or polypeptide. In some examples, thevectors comprise secreted resilin coding sequences that arecodon-optimized for expression in a particular host cell.

As discussed further in the '197 Application, suitable secretion signalsare secretion signals that mediate secretion of polypeptides in therecombinant host cells provided herein. The resilins encoded by thesecreted resilin coding sequences can be further fused to tag peptidesor polypeptides. In some examples, the vectors comprise single secretedresilin coding sequences, while in other examples, the vectors comprise2 or more (e.g., 3, 4, or 5) secreted resilin coding sequences. Thesecreted resilin coding sequences may be identical. Alternatively, atleast 2 of the secreted resilin coding sequences are not identical. Whenat least 2 of the secreted resilin coding sequences are not identical,the at least 2 secreted resilin coding sequences can differ from eachother in the resilins and/or in the secretion signals and/or theoptional tag peptides or polypeptides they encode.

In some examples, the vectors comprise promoters that are operablylinked to the secreted resilin coding sequences such that they drive theexpression of the secreted resilin coding sequences. In furtherexamples, the vectors comprise terminators that are operably linked tothe secreted resilin coding sequences such that they effect terminationof transcription of the secreted resilin coding sequences. In examplesin which the vectors comprise 2 or more resilin coding sequences, the 2or more resilin coding sequences can be operably linked to the samepromoters and/or terminators or to 2 or more different promoters and/orterminators.

The vectors provided can further comprise elements suitable forpropagation of the vectors in recombinant host cells. Non-limitingexamples of such elements include bacterial origins of replication andselection markers (e.g., antibiotic resistance genes, auxotrophicmarkers). Bacterial origins of replication and selection markers areknown in the art. In some embodiments, the selection marker is a drugresistant marker. A drug resistant marker enables cells to detoxify anexogenously added drug that would otherwise kill the cell.

The vectors of the disclosure can further comprise targeting sequencesthat direct integration of the secreted resilin coding sequences tospecific locations in the genome of host cells. Non-limiting examples ofsuch targeting sequences include nucleotide sequences that are identicalto nucleotide sequences present in the genome of a host cell.

Recombinant host cells can comprise the vectors described. In someexamples, the vectors are stably integrated within the genome (e.g., achromosome) of the recombinant host cells, e.g., via homologousrecombination or targeted integration. In other examples, the vectorsare not stably integrated within the genome of the recombinant hostcells but rather are extrachromosomal. Recombinant host cells can be ofmammalian, plant, algae, fungi, or microbe origin. It should beunderstood that the term “recombinant host cell” is intended to refernot only to the particular subject cell but to the progeny of such acell. Because certain modifications may occur in succeeding generationsdue to either mutation or environmental influences, such progeny maynot, in fact, be identical to the parent cell, but is still includedwithin the scope of the term “recombinant host cell” as used herein. Insome examples, the recombinant host cells may comprise geneticmodifications that improve production of the recombinant resilinsprovided herein.

The recombinant host cells are generated by transforming cells ofsuitable origin with vectors. For such transformation, the vectors canbe circularized or be linear. Recombinant host cell transformantscomprising the vectors can be readily identified, e.g., by virtue ofexpressing drug resistance or auxotrophic markers encoded by the vectorsthat permit selection for or against growth of cells, or by other means(e.g., detection of light emitting peptide comprised in vectors,molecular analysis of individual recombinant host cell colonies, e.g.,by restriction enzyme mapping, PCR amplification, or sequence analysisof isolated extrachromosomal vectors or chromosomal integration sites).In some examples, the recombinant host cells provided herein can producehigh titers of the recombinant resilins provided herein.

Production and secretion of recombinant resilins can be influenced bythe number of copies of the secreted resilin coding sequences comprisedin the recombinant host cells and/or the rate of transcription of thesecreted resilin coding sequences comprised in the recombinant hostcells. In some examples, the recombinant host cells comprise a singlesecreted resilin coding sequence. In other examples, the recombinanthost cells comprise 2 or more (e.g., 3, 4, 5, or more) secreted resilincoding sequences. In some examples, the recombinant host cells comprisesecreted resilin coding sequences that can be operably linked to strongpromoters.

The fermentations comprise recombinant host cells and a culture mediumsuitable for growing the recombinant host cells. The fermentations areobtained by culturing the recombinant host cells in culture media thatprovide nutrients needed by the recombinant host cells for cell survivaland/or growth, and for secretion of the recombinant resilins. Suchculture media typically contain an excess carbon source. Non-limitingexamples of suitable carbon sources include monosaccharides,disaccharides, polysaccharides, and combinations thereof. Non-limitingexamples of suitable monosaccharides include glucose, galactose,mannose, fructose, ribose, xylose, arabinose, ribose, and combinationsthereof. Non-limiting examples of suitable disaccharides includesucrose, lactose, maltose, trehalose, cellobiose, and combinationsthereof. Non-limiting examples of suitable polysaccharides includeraffinose, starch, glycogen, glycan, cellulose, chitin, and combinationsthereof. The resulting fermentation can comprise recombinant resilins invarying amounts.

Methods of Producing Recombinant Resilin

The recombinant resilins described herein can be produced according tovarious methods. Such methods are generally performed according toconventional methods well known in the art and as described in variousgeneral and more specific references that are cited and discussedthroughout the '197 Application, unless otherwise indicated. In someembodiments, a method can be utilized to secrete resilin extracellularlyfrom a host cell, which may comprises constructing a vector comprising asecreted resilin coding sequence, transforming the vector into a hostcell, and then culturing the recombinant host cells to secrete resilinextracellularly. The secreted resilin may then be purified, and thepurified resilin can then be cross-linked to form an elastomer. In someexamples, the methods may include the step of transforming cells withvectors provided herein to obtain recombinant host cells providedherein. Methods for transforming cells with vectors are well-known inthe art.

The methods may further include the step of culturing the recombinanthost cells in culture media under conditions suitable for obtaining thefermentations. In some examples, the conditions and culture media aresuitable to facilitate secretion of the recombinant proteins from therecombinant host cells into the culture media. Suitable culture mediafor use in these methods are known in the art, as are suitable cultureconditions.

Purification can occur by a variety of methods known in the art forpurifying secreted proteins from fermentations. Various examples ofcommon steps in such methods include centrifugation (to remove cells)followed by precipitation of the proteins using precipitants or othersuitable cosmotropes (e.g., ammonium sulfate). The precipitated proteincan then be separated from the supernatant by centrifugation, andresuspended in a solvent (e.g., phosphate buffered saline [“PBS”]). Thesuspended protein can be dialyzed to remove the dissolved salts.Additionally, the dialyzed protein can be heated to denature otherproteins, and the denatured proteins can be removed by centrifugation.Optionally, the purified recombinant resilins can be coacervated.Methods of purifying the secreted recombinant proteins from thefermentation can include various centrifugation steps in conjunctionwith solubilizing protein in a whole cell broth or cell pellet withknown chaotropes such as urea or guanidine thiocyanate, examples ofwhich are discussed in greater detail in the '197 Application. Suchmethods and steps, as well as other purification methods are known inthe art and can be used to or adapted to purify resilin, as describedherein. Further detail of one example method can be found in Kim, M.,Elvin, C., Brownlee, A. & Lyons, R. High yield expression of recombinantpro-resilin: Lactose-induced fermentation in E. coli and facilepurification. Protein Expr. Purif. 52, 230-236 (2007). Variousadaptations and combinations of known methods may be made to purifyresilin at scale according to the knowledge of one skilled in the art.Examples of specific solutions and solvents for cross-linking, andvarious specific combinations thereof are discussed above and in the'197 Application.

Overview of Example Products Using the Resilin Material

In some embodiments, the cross-linked resilin compositions describedherein can be used to provide a composition having improved physicalproperties, including, e.g., for absorption of energy from an appliedforce as desired. In some embodiments, the cross-linked resilincompositions described herein can be used to replace rubber or othersynthetic elastomers in existing products. In particular, some of thecross-linked resilin compositions provided herein can absorb a largeamount of force, while not transitioning to an inelastic material.

In some embodiments, the cross-linked resilin compositions providedherein can be used as an outer for a shoe, including at least forportions of the midsole. In some embodiments, the cross-linked resilincompositions provided herein can be used as part of a core for a golfball, softball, or the like. In other embodiments, the cross-linkedresilin compositions provided herein can be used in handles or grips,e.g., for sports equipment such as golf clubs or tennis rackets, asbicycle grips or motorcycle grips, or as grips for tools and industrialuses such as hammers, nail guns, jackhammers, and any other tools whereit is preferable to absorb and return energy. In some embodiments, thecross-linked resilin compositions provided herein can be used inbrushings or dampenings, e.g., skate board trucks or hard drive plattervibration dampener. In some embodiments, the cross-linked resilincompositions can be used as material for wheels, such as for skateboards, roller blades, or scooters. In some embodiments, thecross-linked resilin compositions provided herein can be used for safetyand protective gear, such as padding for protective equipment such ashelmets, elbow or knee pads, shoulder pads, protective gloves, or hardhats, or as a protective outer layer to protect the skin from abrasions.

In some embodiments, the cross-linked resilin compositions providedherein can be used for automotive parts, e.g., suspension componentssuch as bushings or shock absorbers, or for interior cushioning such asseat bolsters and lumbar support. In some embodiments, the cross-linkedresilin compositions provided herein can be used for tires and innertubes. In some embodiments, the cross-linked resilin compositionsprovided herein can be used for suberballs. In some embodiments, thecross-linked resilin compositions provided herein can be used for shoeinsoles, midsoles, and outsoles. In some embodiments, the cross-linkedresilin compositions provided herein can be used in a padded mat. Insome embodiments, the cross-linked resilin compositions provided hereincan be used for several types of gaskets or O-rings. In someembodiments, the cross-linked resilin compositions provided herein canbe added to plastic items to increase their impact resistance. In someembodiments, the cross-linked resilin compositions provided herein canbe used for protective cases, such as phone or tablet cases. In someembodiments, the cross-linked resilin compositions provided herein canbe used for rubber stamps. In some embodiments, the cross-linked resilincompositions provided herein can be used for rollers. In someembodiments, the cross-linked resilin compositions provided herein canbe used for rubber bands.

In some embodiments, the cross-linked resilin compositions providedherein can be used for shoe soles, basement flooring, noise protectionfor sound studios, car bumpers, cushion pads, door mats, yoga mats, drumpads, window wipers, car tires, fire hoses, electrical wiringinsulation, rubber bands, rubber ducks, elastic gloves, cookingutensils, rain boots, teething toys, bicycle tires, watches, jars,gaskets, hair ties, flip-flops, phone cases, medicine balls, bouncyballs, seals for electronic devices to prevent contamination from wateror dust, refrigerator or freezer door seals, seals to prevent air flowin or out of a chamber, trampolines, pacifiers, window seals, Halloweenmasks, garden hoses, table tennis rackets, conveyer belts, ducting,stamps, or balloons.

It is to be appreciated that the above-described fermentation,purification, cross-linking, and solvent-exchange steps, in variousexamples (including those discussed specifically above and those thatmay be apparent or derived based on the above description) are derivedor adapted to produce a resilin-based material generally resemblingvarious elastomers. To that end, such processing steps can beparticularly applied to produce specific resilin-based materials havingcharacteristics or properties (including tactile, visual, and physical,as described in greater detail herein) similar to those of variouselastomers, including elastomers of various types or having variousknown properties or attributes. In this manner, resilin-based materialcan be fermented, purified, cross-linked, solvent exchanged, orsubjected to various post-processing steps according to the processesand variations described herein and in various combinations to produceraw-material that can be manufactured or fabricated into differentproducts typically, or in various forms, being primarily of, orotherwise featuring or including, an elastomer. In certain forms andcompositions, this resilin based material may result in products orarticles that meet or exceed consumer, retailer, or manufacturerexpectations for similar products of or including elastomer. In thismanner, such products comprised of, using, or incorporating the varioustypes of resilin material that may be produced according to or in lightof the above description may provide benefits to the consumer andmanufacturer beyond what is possible with traditional elastomers and inaddition to the ecological, environmental, and humanitarian benefitsthat may be realized by substituting the resilin materials describedherein for leather.

Footwear Including Resilin Compositions

In accordance with the preceding description, in one example, theresilin material described herein can be incorporated into in varioustypes and forms of footwear, including in any of the various portions offootwear (among the various types thereof discussed herein and thatwould be understood based on the description herein) that can be, ortypically are, formed of elastomeric material, including in substitutionfor various types of petroleum-based elastomers (e.g., ethyl vinylacetate (“EVA”)). In various forms, the resilin material describedherein can be used for all or portions of a shoe “outer” for many typesof shoes, as well as various portions of a shoe upper or, for some typesof footwear, the entire upper. In such instances, specificimplementations of the resilin compositions described herein can be usedto derive materials having appropriate characteristics and can be usedfor the various elastomeric portions of an article of footwear with suchresilin materials being fabricated or manufactured into the desiredform, according to the particular footwear portion or component,various, non-limiting examples of which are discussed herein.

Referring to the embodiment illustrated in FIG. 1, reference numeral 10generally designates a shoe, particularly in the form of a sneaker. Asdiscussed herein, the term “sneaker”, when used in reference to a typeof footwear, connotes a style and construction capable of many practicalvariations, including with respect to particular stylisticimplementations thereof and the particular construction within agenerally accepted framework. Still further, sneakers can be designedand constructed for different types of activities or use, with varioustypes of sneakers exhibiting ranges of stylistic or functionalversatility making them suited for certain ranges of activities and useof varying scope.

In this respect, the shoe 10 illustrated in FIG. 1 may be characterizedas an “athletic” sneaker, wherein the use of the term “athletic” inconnection with the term “sneaker” to describe the depicted style offootwear does not imply or require that such footwear be strictly usedor otherwise useable for any specific type of athletic activity, or forany athletics at all. In one example, an article of footwear may simplybe of the style or construction of or evoking athletic footwear so as toencompass such footwear, whether used or intended for athletic activityor not (e.g., “lifestyle”, “athleisure”, or fashion-footwear styled asor similar to athletic sneakers or other variations of athleticfootwear, as described below). Further, the descriptions made herein,including in reference to the drawing figures, are merely exemplary withrespect to the footwear described and illustrated, so that variationsmay be made to the footwear described herein for purposes of style orfit and/or to make footwear based on the principles and constructiondescribed herein suitable for various purposes or conditions. Evenfurther, although construction and production techniques may bediscussed herein with respect to particular styles of footwear (e.g.athletic sneakers), such construction and production techniquesdiscussed with respect to one type of footwear may be an acceptablealternative for comparable construction and production techniquesdiscussed herein with respect to other types of footwear (e.g., hikingboots, sandals (including sport sandals), athleisure, lifestyle, and thelike).

Continuing with reference to FIG. 1, the illustrated sneaker 10 isexemplary of typical construction of sneakers and includes an upper 12,a midsole 14, and an outsole 16, with the upper 12 defining an interior18 generally suited for receiving the foot of a wearer, and the outsole16 forming the portion of the sneaker 12 contacting the ground beneaththe foot of the wearer. In this respect, the construction of thedepicted sneaker 10 is generally typical of other types of footwear withit being noted that the combined midsole 14 and outsole 16 may becollectively referred to as the footwear “outer” and may be used invarious forms other than the depicted midsole 14 and outsole 16. In oneexample, an outer may consist of a midsole of resilin material thatexhibits acceptable abrasion resistance such that at least portions ofthe ground-contacting surface typically included in a separate outsolemay be formed in the midsole of the resilin material, as discussedfurther below. As shown in the example of FIG. 1, the midsole 14 ispositioned between the upper 12 and the outsole 16 and provides supportand cushioning for the sole of the foot, particularly during impact withthe ground, as made by the outsole 16.

As can be seen in FIG. 2, the interior 18 of the upper 12 is generallyenclosed at the lower portion thereof by a lasting board 24 to which theupper 12 is affixed around or adjacent a lower perimeter 22 of the upper12 (depending on the particular construction method, as discussedfurther below). The lasting board 24 and/or the portions of upper 12adjacent perimeter 22 are, in turn affixed with midsole 14 with thelasting board 24 being positioned above the midsole 14. An insole 24(FIG. 3) may be placed within the interior 18 above the lasting board24. The insole 20 may be at least somewhat cushioned to provideadditional comfort to the user and to cover the stitching used to attachthe lasting board 24 around the perimeter 22. In one aspect, the insole20 may also include or be completely of a resilin material, as describedherein and exhibiting the desired energy absorption and/or tactilequalities. This may be done by fabricating the insole 20 entirely fromthe resilin material or by covering a resilin cushioning layer with athin layer of fabric, leather, mycelium material, or the like such thatthe resilin material provides a cushioning layer for the insole 20.Still further, the insole 20 may be of a coated resilin material,according to various examples discussed below.

As can be seen in FIGS. 1 and 2, the presently described sneaker 10 isexemplary of a sneaker, particularly the upper 12, manufactured using a“cut and sew” process by which the upper 12 is fabricated from a numberof individual sections of stock material corresponding with variousportions of the upper 12. In particular, the individual sections are cutfrom the stock material in flat, two-dimensional shapes, as needed asdictated by the desired final form of the upper 12, and are sewntogether along various seams that at least partially give the upper 12its desired three-dimensional form. Such sewing may be augmented by theuse of various adhesives along the seams and may be carried in whole orin part over a last that corresponds with the desired shape of theinterior 18 of upper 12. In particular, the lasting board 24 istypically sewn to upper 12 over a last and, with respect to typicalconstruction of the depicted athletic sneaker 10, and similar footwear,completed using a “Strobel” stitch using specialized machinery thatjoins the material portions of the upper 12 that define the perimeter 22with lasting board 24 in an abutting edge-to-edge seam. The resulting“Strobel sock” including the assembled upper 12 and lasting board 24 isthen affixed with the midsole 14, which is most often done usingadhesive or the like. In some forms of construction, the affixationbetween the lasting board 24 and the midsole 14 can be augmented orcompleted using stitching, such as Blake stitching or the like, or usingstitches along particular areas of the upper 12 associated with featuresattached to the midsole 14, as discussed further below.

As can be appreciated, the pieces and sections of upper 12 may generallycorrespond with particular areas of the upper 12 but may vary accordingto their particular shape and placement depending on the desiredstylistic appearance of the sneaker 10, as well as the desired fit,flexibility, and support of the athletic sneaker 10 (which may beinfluenced or dictated by the intended use of the athletic sneaker). Inthe exemplary depiction of FIGS. 1 and 2, the various portions of theupper 12 may include a toe tip 26, and a vamp 28 extending from the toetip 26 upward to the throat 30 of the athletic sneaker 10. A tongue 32extends upwardly along the throat 30 from vamp 28, and opposite quarters34 extend rearwardly from the toe tip 26, to define the portions oflower perimeter 22 along the respective sides of upper 20, anddownwardly away from the throat 30. A heel counter 36 extends around therear of the upper to connect between the two quarters 34 a,34 b aroundthe heel of the wearer. Further, medial and lateral collar portions 38can extend upwardly from heel counter 36 and rearwardly from therespective medial and lateral quarters 34 to define respective portionsof the topline 40 of the upper 12. A heel tab 42 is positioned above theheel counter and connects between the rearward-most ends of therespective collars 38 to define the rear section of the topline 40. Aninner liner 44 (FIG. 3) can extend through all or part of the upper 12to define the interior 18 thereof and can be affixed with the individualouter portions of the upper 12 along which it extends.

In a similar manner the outer, including the presently-depicted midsole14 and outsole 16 can include a number of different regions that may bedefined relative to one another to varying degrees or by varyingcharacteristics. Most notably, the outer (midsole 14 and outsole 16) canbe structured and defined by the portions of the foot that it supportsrelative to the ground and the manner by which such support is achieved.In this respect, both the midsole 14 and outer 16, where applicable, canbe discussed in terms of corresponding heel (or rear-foot) 52 portions,mid-foot 54, and fore-foot 56 portions, as well as medial 58 and lateral60 portions. In various contexts, combinations of such portions can beused to refer with greater or lesser specificity to the portions of theouter, for example, by reference to the medial, 58 heel 52, or the like.Still further, various specific portions within the various regions maybe of specific relevance and, accordingly, given specific designations,including the heel-strike 62, the arch 64, the metatarsal head area 66(i.e. beneath the balls of the feet), and the toe spring 68. Both themidsole 14 and the outsole 16 can be of varying materials, shapes,constructions, etc. within and among these regions, sub-regions, orspecific areas to provide the desired fit, cushioning, stability,traction, and aesthetic qualities of the particular type or specificimplementation of an article of footwear, such as the depicted sneaker10, as well as to achieve any desired weight characteristics of thesneaker 10, various examples of which are discussed below.

In general, the midsole 14 can be particularly structured toanatomically correspond with the sole of a wearer's foot (or a range ofwearers' feet according to known schemes for sizing sneakers and thelike). Such structuring may include constructing midsole 14 with agreater thickness in the heel 52 portion and a comparatively lowerthickness in the fore-foot 56, which can provide increased materialcushioning for the heel 52, as the heel 52 typically makes first contactwith the ground during a normal stride with a forward-leaning footposition and reduced material under the fore-foot 56 for weight-savingpurposes, as less cushioning is needed under the fore-foot of thewearer. Still further, the heel strike 62 portion may be shaped orotherwise structured to increase cushioning even further in such ahigh-impact area, as well as to promote forward-rolling of the foot tosmoothly bring the fore-foot 56 into contact with the ground. In asimilar manner, the toe spring 68 can be upturned or otherwisestructured to reduce effort in push-off by the wearer, such as duringrunning or walking.

Within the mid-foot 54, the midsole 14 can be structured to providereduced weight by removing material in areas where the foot does notnormally contact the ground. In this manner, the midsole 14 can bestructured to extend upwardly relative to the heel 52 and fore-foot 56areas, at least on the medial side 58 thereof, with the midsole 14remaining close to the ground plane within the lateral side 60 in someapplications. The outsole 16 can be structured to correspond with suchmidsole 14 construction, including by eliminating material within themedial 58 mid-foot 54 or by being formed in separate heel and forefootportions with no portion of outsole 16 being positioned along themid-foot 54, as shown in FIG. 3. As discussed further herein, theoutsole 16 can be further structured, including by incorporation ofdifferent materials as well as within other portions thereofcorresponding to the various regions and areas discussed above. Themidsole 14 can be further structured within the mid-foot 54 such thatthe arch 64 extends upwardly to provide support for the arch of thewearer to relieve muscle strain during heel-to-toe rolling of the footand during push-off in particular. In this respect, the midsole 14 maynot fully extend upwardly through the entire arch of the wearer (i.e.,along the side of the foot), with portions of the upper 12 and theinsole 20 (and/or specific inserts) providing additional support for thearch of the foot extending upwardly and medially from the midsole 14.

As shown in the exploded view of FIG. 3, midsole 14 can be fabricatedfrom a number of different pieces of the resilin material using selectedones of the resilin compositions described above, to achieve animplementation of the above-described structure. In the illustratedexample, a lower midsole 14 a can extend over the entire length of thesneaker 10, including through the heel 52, mid-foot 54, and fore-foot56. An intermediate midsole 14 b can, similarly, extend over the entirelower midsole 14 a and can provide different properties with respect tocushioning or appearance to contrast with lower midsole 14 a. Thecombined lower midsole 14 a and intermediate midsole 14 b can provide atleast the desired cushioning and positioning for fore-foot 66. A heelwedge 14 c can be positioned over intermediate midsole 14 b and canextend generally through the heel 52 portion of the midsole 14. In thisrespect, the heel wedge 14 c can be tapered to a thin edge within themid-foot 54 or fore-foot 56 portions to provide a smooth transition tothe intermediate midsole 14 b. Further, the heel wedge 14 c can bestructured to extend upward along at least the medial 58 portion of themid-foot 56 to provide the desired support for at least a portion of thearch 64. In this respect the heel wedge 14 c can be asymmetrical toextend farther into the mid-foot 56 along the medial 58 side tocorrespond with the positioning of arch 64, as well as the generalasymmetry of the foot.

As can be appreciated, the midsole 14 construction depicted in FIGS. 1-3is similar to what may be referred to as a “retro” style sneaker 10,wherein midsole 14 is constructed by a “cut-and-buff” process in whichlarge sheets of bulk polymer foam, such as EVA, are cut into the desiredshapes for lower midsole 14 a, intermediate midsole 14 b, and heel wedge14 c, which are then cemented together using suitable adhesive. Exposededges, as well as the toe spring and heel kick areas, of the resultingassembly are then “buffed” using an abrasive material on appropriatemachinery to form a consistent outer edge in the desired final shape. Inthis manner, the separate pieces 14 a,14 b,14 c of the midsole 14 appearas a cohesive unit, while utilizing the laminated structure to achievethe desired shape profile and cushioning characteristics.

The presently-described resilin material can be used in variousimplementations to achieve a midsole of the depicted retro style. In oneexample, the resilin material can be formed, particularly by theabove-described cross-linking and solvent exchange steps, in sheetscorresponding to the desired maximum thicknesses for the lower midsole14 a, intermediate midsole 14 b, and heel wedge 14 c (noting that insome instances, the lower midsole 14 a and the heel wedge 14 c may be ofthe same stock sheet). Additionally, as it is typical to use variousfoams (e.g., EVA foam) in one or all portions of a shoe midsole 14, theresilin material used for the present midsole 14 (or at least one of theresilin materials in examples where different materials are used for thevarious portions 14 a,14 b,14 c of the midsole 14) can be a foamedresilin material, as discussed in general above. The desired lowermidsole 14 a, and intermediate midsole 14 b can be cut into the desiredprofile (depending on the size, style, and overall desiredconfiguration) of midsole 14, and the heel wedge 14 c can be cut in boththe desired profile (i.e. matching the profile of lower and intermediatemidsoles 14 a,14 b within the heel area 52) and angled wedge shape.

In the alternative, the individual pieces 14 a,14 b,14 c can be directlymolded into the above-described shapes, such that additional cutting orshaping is not needed such that the appearance of a cut-and-buff midsoleis achieved without the wasted material from the actual process. In oneexample, such molding can be achieved, as discussed above, by providingmolds with cavities corresponding to the desired shapes for the midsolepieces 14 a,14 b,14 c and filling such cavities with a purified anddenatured recombinant resilin composition, as discussed above, with across-linking solution of the various types discussed above such thatthe resilin protein cross-links in the mold. In this respect, the massesof cross-linked resilin material will generally retain the shape derivedby the mold cavity. One of the various nonaqueous solvents can beexchanged for the solvent originally present in the resilin materialmass to achieve the desired composition for the midsole pieces 14 a,14b,14 c. It is noted that the solvent exchange process may result in someshrinking of the molded resilin material pieces (e.g., 14 a,14 b,14 c),which may be in the range of between 10% and 40% and in one embodimentabout 25% of the pre-exchanged resilin material volume. In this respect,the molding step may be carried out to account for a determined orpredicted level of shrinking, including by appropriately adjusting thesize and configuration of the corresponding mold cavity.

In various implementations of either of the above-described fabricationprocesses for midsole 14, different recombinant resilins or differentmixtures of different recombinant resilin compounds, as well asdifferent cross-linking solutions and different nonaqueous solvents canbe used to achieve different midsole pieces 14 a,14 b,14 c withdifferent properties determined to be desirable for its overallincorporation into midsole 14. In one respect, the composition of lowermidsole 14 a can be selected to provide a desired level of energy returnand/or resilience, while heel wedge 14 c and/or intermediate midsole 14b can be selected to provide a desired level of cushioning. Additionallyor alternatively, the particular characteristics of foamed resilin canbe specifically derived or controlled to achieve additionalcharacteristics advantageous to the various portions 14 a,14 b,14 c ofmidsole 14 and/or of midsole 14 as a whole. In one aspect, theintroduction of bubbles to derive a foamed resilin material can reducethe specific gravity of the material and can, accordingly, be used,subject to other requirements or desired characteristics of midsole 14to reduce the weight thereof by, effectively, reducing the overallamount of material. Additionally, the introduction of distributed airbubbles throughout a matrix of resilin material can alter the materialproperties of the overall solid beyond the simple reduction in specificgravity. In one aspect, the presence of air bubbles can change theresponse of the midsole 14 to the application of compressive loads dueto the foamed solid exhibiting a two-stage compressive durometerresponse. In this respect, the foamed solid resilin material can exhibita first compressive durometer response as the air bubbles collapse underload, such response being dictated by the rate at which air exits thevoids and from the foam solid overall (in the case of open-cell foam,which results from the above-described step of air bubble introduction).A second response is presented once all or a significant number of thecells are collapsed such that the mass becomes more solid and exhibitsan increased durometer more comparable to that of the material itself,without the presence of voids.

In various examples, the different methods for introducing air bubbles,as well as the use of different types and quantities of additives,specific crosslinking processes or agents, and the like, can result infoams having varying cell sizes and, accordingly different properties ofthe resulting foamed resilin solid. In one example, fumed silica, as athickening agent, and sodium bicarbonate, as a chemical blowing agent,can be added to the resilin before crosslinking. These additives canfacilitate uniform distribution of air bubbles during the introductionprocess, resulting in more uniform cell distribution in the finishedfoamed resilin solid. Altering the amount of the fumed silica and/orsodium bicarbonate can allow for one mode of control for the averagebubble size within the resilin material and can promote evendistribution of such air bubbles.

In one implementation, midsole 14 can include at least one portion(including portions 14 a, 14 b, and 14 c) of a solid resilin foamprepared by dissolving the purified recombinant resilin material in PBSat a pH of about 7.4. Sodium bicarbonate can be added to the resilinsolution as a chemical blowing agent, which may be done in combinationwith fumed silica, as a thickening agent in varying amounts, to controlthe size and distribution of air bubbles, as discussed above. In variousexamples, between 6 mg/mL and 20 mg/mL of sodium bicarbonate (or between0.2% and 2% by weight) can be added to the resilin solution. In general,sodium bicarbonate may be added at a maximum amount of about 33 mg/mL toprevent the sodium bicarbonate from inhibiting gelation. The furtheraddition of fumed silica (or other thickening agent) in quantitiesranging between 4% and 10% by weight may result in a resilin solid(i.e., after the subsequent processing steps with the addition offurther components as discussed below) with generally evenly-distributedbubbles. In specific implementations, the addition of fumed silica inrelatively low amounts, including between 4% and 5% by weight, result inrelatively large bubbles, in the range of about 0.2 mm to 2 mm, withinthe resulting resilin material. Adding fumed silica in an amount greaterthan 5% by weight (including in an amount of up to about 10% by weight)may result in a foamed resilin with relatively smaller bubbles (andincreased overall density), including bubbles in the range of 0.05 mm to0.2 mm.

A solution of ammonium persulfate in a concentration of between 100 mMand 225 mM can be added to the purified and thickened resilin. Xanthangum may also be used in combination with the ammonium persulfatesolution to further increase the viscosity over the thickening agent tofurther aide in trapping bubbles within the solution. In variousspecific implementations, ammonium persulfate can be added in amountsbetween about 10% and 20% by weight and, more specifically in amountsbetween 13% and 16% by weight. In such implementations, xanthan gum canbe added in amounts between 1% and 2%. A crosslinking catalyst can beadded before or after addition of the foaming agent and can be selectedto be activated by one of heat or light. In one example ruthenium (II),which is activated by white light, can be used in amounts between 10%and 20% by weight (other catalysts can be used as an alternative,according to the examples discussed above). After addition of thecatalyst and foaming agent, the solution can be vortexed to allowbubbles to develop within the resilin material. In one example,vortexing can be carried out for 3.5 hours at a temperature of about 80°F. After the solution is vortexed, it can be appropriately crosslinked(e.g., using heat or light) and can be subjected to a solvent-exchangeprocess, as discussed above, including using propylene glycol in anexample. Examples of foamed resilin material solids S1 and S2 with opencells C1 and C2 or relatively smaller and relatively larger sizes,respectively, dispersed in a resilin matrix M1 and M2 are shown in FIG.4.

In additional or alternative implementations, a syringe pump can be usedto introduce dissolved nitrogen, while crosslinking the resilin, aseither a primary source of air bubbles within the resilin material, or asupplement to air bubble generated using an agent, as discussed above.In one implementation, an ammonium persulfate solution (e.g. of either225 or 550 mM) can be added to a solution of resilin in PBS (e.g. ofabout 27% by weight of resilin). The resulting solution can then becentrifuged (e.g. for about 5 min at about 7200 rcf) and added to asyringe pump. The pump can then be purged with nitrogen gas (e.g. forabout 3 minutes) and then sealed. The pump can then be set to either 500psi or 1600 psi and heated (e.g. for between about 2 hours and about 6hours at about 83° C.). After releasing the pressure, the resilin can beheated for an additional interval (e.g. for between about 1 hour andabout 2 hours at about 83° C.) It is noted that crosslinking at thesepressures may proceed at a slower rate than crosslinking at atmosphericpressure, likely due to the increased amounts of dissolved oxygen. Theaddition of a thickener, as discussed above may further enable uniformfoams via such a process. The various foamed resilin materials can alsobe used in the additional products discussed above, including in coresfor golf balls, softballs, or the like, handles or grips, e.g., forsports equipment such as golf clubs or tennis rackets, as bicycle gripsor motorcycle grips, or as grips for tools and industrial uses such ashammers, nail guns, jackhammers, etc. In further embodiments, the foamedresilin materials provided herein can be used in safety and protectivegear, such as padding for protective equipment such as helmets, elbow orknee pads, shoulder pads, protective gloves, or hard hats.

The separate pieces 14 a,14 b,14 c of midsole 14, derived by any of theprocesses discussed above, including those that result in a foamedresilin material having cells of varying sizes and correspondingdensities, can then be laminated together using appropriate adhesives,adhesion of which with the present resilin material being, in someinstances, augmented by the prior application of a primer material, suchas acetone or a similar material. In the alternative, it may be possibleto affix together multiple pieces of the present resilin material usingan additional cross-linking step prior to solvent exchange, wherein alayer of the cross-linking solution is applied between pieces, such asthe midsole pieces 14 a,14 b,14 c of resilin material, which are thenretained in contact with each other. The addition of heat and/or light(depending on the particular cross-linking solution) can help achievethe desired cross-linking of the proteins along the contact area betweensuch pieces. After such cross-linking, the pieces 14 a,14 b,14 c arejoined together and the solvent exchange step can be carried out.

In a still-further variation of the above molding process, it may bepossible to add different purified and denatured recombinant resilincompositions into a single mold in layers corresponding with differentportions of midsole 14, including the pieces 14 a,14 b,14 c illustratedin the present example and to apply a single cross-linking solution tothe layered materials to obtain a single midsole 14 having multipledifferent types of resilin material in layers or other portions therein,which can then be subjected to solvent exchange to obtain a midsole 14having different properties in different areas thereof, including in theillustrated layers. Such a process may be used to obtain othervariations of material in connection with different midsole 14 typesdiscussed below.

In some examples, the depicted layered structure can include layers 14a,14 b,14 c having different configurations and corresponding propertiesaccording to the above descriptions. In particular, the different layers14 a,14 b,14 c can be of foamed resilin materials having different cellsizes and/or densities. In one example, the outermost layer 14 a can beof a higher density configured for protection and energy return with atleast the middle layer 14 b having a lower density for cushioning. Otherarrangements, including various combinations of foamed and non-foamedresilin material can be implemented according to the desiredcharacteristics of the midsole 14. As can be appreciated, a foamedresilin material according to the above description can also be used forall or a part of insole 20 (FIG. 3) and for various padding or the likein upper 12 (including within collar 38 and tongue 32).

In additional examples, the general effects (reduced specific gravity,two-stage durometer response, etc.) of a foamed resilin material can beachieved by removing material from solid resilin material layers priorto laminating, or adhering, them together. In one example several sheets(including more than the three depicted layers 14 a,14 b,14 c) can bemade in various thicknesses, including in one example thicknesses ofbetween about 1 mm and 5 mm. The sheets can be cut in various patternsto remove material with a mismatch in the patterns among the variouslayers to mimic the distribution of cells in a foamed material afterlamination. In various examples, the sheets can be cut using a die,water jet, lasers, etc. in either stocked patterns or in the desiredshape of midsole 14. In the latter example, the patterns can be variedand scaled according to the midsole size or configuration, which can bedone to ensure that no holes intersect the edge of the midsole 14 and/orto achieve a midsole 14 with varying density characteristics in thevarious regions thereof. Additionally, a solid layer can be provided asthe outermost layer to provide a closed, smooth outer surface formidsole 14, including for adhesion with outsole 16. The layers can beadhered together or can be re-crosslinked after arrangement in thedesired configuration. Still further, the layers can be sublayers, suchthat they can be arranged in different overall layers, such as thedepicted layers 14 a,14 b,14 c having different properties.

One example, of a layered structure is shown in FIGS. 5A and 5B, whereinthree sublayers 14 a ₁, 14 a ₂, and 14 a ₃ are shown having holes, orperforations 96, therein. As shown, in FIG. 5A, the outermost sublayers14 a ₁ and 14 a ₃ have perforations 96 that are offset from theperforations 96 in the middle sublayer 14 a ₂. As shown in FIG. 5B, whenthe sublayers 14 a ₁,14 a ₂,14 a ₃ are stacked, the perforations 96 inthe outer sublayers 14 a ₁ and 14 a ₃ align with each other in an offsetmanner from the perforations 96 in the middle sublayer 14 a ₂. Thedepicted pattern can be repeated in additional sublayers to provide amidsole layer, such as layer 14 a of the desired thickness. In a furtherexample, an entire midsole 14 can be of such a construction. As shown inFIG. 6, the perforations can vary in size including between regions ofthe midsole. In one implementations, the relatively smaller perforations96 a can be positioned to provide a denser portion of the midsole 14that can, for example, be positioned in the arch area 64 of midsole 14.The relatively larger perforations 96 b can be positioned to provide arelatively softer portion of the midsole 14 that can, for example, bepositioned in the heel strike area 62 of the midsole 14. Other examplesand arrangements are possible according to known midsole constructionsand the additional examples provided herein. In a further example shownin FIGS. 7A and 7B, holes in various geometric shapes other than circlesor standard perforations can be used. Such shapes can be irregular oruniform and/or can vary with different regions of the midsole 14 andmay, in some implementations be derived to result in a so-calledmacrostructure based on the various arrangements of openings 98 amonglayers 14 a ₁,14 a ₂,14 a ₃ (FIG. 7A) when laminated together (FIG. 7B).Such structures can exhibit asymmetric properties during loading,including an asymmetric, or inversed, Poisson's ratio during loading orwhen subject to stress such that certain portions may contract in onedirection (i.e. along X, Y, or Z axes) when loaded (i.e. in another ofthe X, Y, or Z axes) or may otherwise increase in durometer in responseto loads. Such properties, in some implementations, can be tuned to theneeds of the specific midsole 14 and/or specific areas thereof.

Continuing with respect to FIG. 3, the above-described layered structurefor midsole 14 can also provide for the incorporation of an internalshank 70 therein. In general, shanks, such as the depicted,substantially flat “credit card” shank 70, help to provide structuralrigidity to a midsole 14 through the mid-foot 54, where the wearer'sfoot exhibits some flexibility (i.e., more than in the heel 52) suchthat many potential wearers experience strain or fatigue within theplantar muscles of the foot and the adjacent soft tissue throughrepeated loading and unloading within the mid-foot 54, such as duringrunning or rapid changes in lateral direction. In this respect, therigidity provided by the shank 70 localizes flexing of midsole 14 towithin the fore-foot 56, where the joints of the foot are most flexible,while also maintaining the insole 20 in more consistent contact with thewearer's foot through the mid-foot area 54 to reduce muscle strain andprovide increased support for the foot within the arch 64. In theillustrated example, the shank 70 is of a generally flat piece ofmaterial (that may be ribbed or otherwise shaped to increase rigiditywhile maintaining an overall flat character) with high stiffness andhigh elastic deformation such as various plastics, carbon fiberreinforced polymer (“CFRP”), Kevlar® reinforced polymer, steel (such asspring steel), or the like. Such a shank 70 can be laminated between thelayers 14 a,14 b,14 c of the midsole 14, including between the lowermidsole 14 a and the intermediate midsole 14 b, as illustrated. Asfurther shown, the shank 70 can be inserted in a cavity 72 formed inlower midsole 14 a, such as by additional processing of a cut resilinsheet (e.g., grinding or machining) or during the above-describedmolding process.

The above-described midsole 14, fabricated according to any of thevarious examples discussed above and further variations that would beunderstood based on the description and depiction of the midsole inFIGS. 1-3, can be bonded with outsole 16, including using variouscements or adhesives used for such purposes in connection with midsoles14 of a typical foam construction. In some examples, the surface ofmidsole 14 (at least in areas where outsole 16 is present) may betreated with a primer material prior to application of the selectedcement or adhesive to improve bonding, depending on the particularcomposition of the resilin material of midsole 14. Solvent-basedadhesives (also referred to as cements) have been used for suchpurposes, including in affixing midsole 14 to upper 12, and aregenerally accepted as having a relatively low cost and rapid fixingtimes and high workability. Such solvent-based adhesives and cements canbe used with parts or portions of the sneaker 10 of the present resilinmaterial in the same way that they can be used with elastomers,including to affix outsole 16 to midsole 14 and to affix midsole 14 toupper 12, as well as to attach together various portions of midsole 14formed of different pieces of resilin material. More particularly, suchadhesives can be used to affix the outsole 16 to the midsole 14 or toaffix additional elements with upper 12, including the depicted heelstabilizer 62, which is fixed between the rear portions of both theupper 12 and lasting board 24 and the midsole 14.

In some circumstances, ultraviolet (“UV”) light curing or activatedadhesives can be used to replace solvent-based adhesives in whole or inpart. Such UV curing or UV activated adhesives can include acrylic-basedcements or modified epoxy materials. In either case, the compoundincludes a photoinitiator that undergoes a chemical reaction whenexposed to UV light, causing the release of byproducts to that reaction.Those byproducts interact with the remaining compound to cause hardeningof the compound or to initiate the reaction that results in hardening.The incorporation of and reliance on the photoinitiator allows for thecement or adhesive to cure “on demand” rather than within a shortinterval from application (e.g. exposure to air in an acrylic cement ormixing in the case of an epoxy). This may allow for the various portionsof upper 12 and/or midsole 14 to be coated with the adhesive with eachsuch piece being activated when ready for affixing with the desiredother piece or element. Various heat-activated adhesives can be used ina similar manner. In general, such adhesives can be made to set upon theapplication of heat above a certain threshold temperature or can useheat as a catalyst for curing (in the case of epoxy, for example). Inone example, the heat-activated adhesive can be applied, as desired,with the assembled sneaker 10 being subsequently run through a heattunnel to initiate or exacerbate the setting of the adhesive to resultin the finished component or product. In some applications, theadhesives can exhibit relatively lower levels of adhesion in an initialstate such that pieces or components can be assembled without stitchingbefore heat is applied to set the heat-activated adhesive.

Still further, water-based adhesives and cements have been developed toact as a replacement for solvent-based compounds, as solvents frequentlyinclude volatile organic compounds (“VOCs”) or other polluting chemicals(that may also be flammable). In one example, a polyurethane adhesive,for example, may have water as its primary “solvent” in that setting ofthe adhesive requires that the water evaporate from the compound.Accordingly, the application of heat may be used to speed or cause theadhesive to set. Additionally, pre-heating of the material to be affixedcan also help speed the setting process. Water-based adhesives mayprovide certain characteristics that make them advantageous for the usein shoe fabrication, including fabrication of the present sneaker 10.Water based adhesives can exhibit reduced stiffening of the material andcan be made of a relatively high viscosity to prevent absorption intothe materials prior to setting, while still being sufficientlysprayable. Accordingly, in the same manner discussed above, water-basedadhesives can be used to affix elements to upper 12 or to fix the upper12 and lasting board 24 with the midsole 14.

As also mentioned above, the outsole 16 may be formed of one or moreportions of rubber (including various synthetic rubbers and the like)selected for desirable characteristics, including density, abrasionresistance, bonding ability, and the like. In some implementations, theoutsole 16 can also be of a resilin material that may be producedaccording to selected variations of the above-described process, toachieve desired density and abrasion resistance similar to what may bedesired of rubber (including synthetic rubber). In such an example, theoutsole 16 can be molded of resilin material, as discussed above, toachieve the desired ground-contact pattern, which may vary based on theintended use of sneaker 10. As discussed above, the outsole 16 may beapplied over the entire outer surface 74 of midsole 14 (i.e., thesurface closest to the ground). In a variation of the illustration inFIGS. 1-3, the outsole 16 may comprise a number of pieces of outsolematerial (including of varying characteristics) that can be affixed withmidsole 14 in areas where contact with the ground is made and/or wheregrip or durability is desired.

Returning to FIGS. 1 and 2, opposite outsole 16, midsole 14 can beaffixed with upper 12. As discussed above, such bonding may also beachieved using cement or adhesive, selected to achieve the desiredbonding with upper 12 and midsole, one or both of which may bepre-treated with a primer or the like prior to cement or adhesiveapplication. As illustrated, the upper 12 of the present example is of aStrobel construction, such that midsole 14 is primarily bonded to thelasting board 24, as well as the adjacent portions of the perimeter 22of the exposed portion of upper 12 (defined around the various portionsthereof discussed above). As shown, a heel stabilizer 74 can beassembled between midsole 14 and upper 12 along the perimeter of theheel 52 with portions thereof extending respectively upward and downwardalong adjacent portions of the midsole 14 and heel counter 36. Heelstabilizer 74 can add structure and protection in the area round thewearer's heel and can improve adhesion between midsole 14 and upper 12around the heel 52 by being cemented with both upper 12 and midsole 14,both in the area directly between midsole 14 and upper 12, as well asalong the side surfaces thereof in the areas contacted by stabilizer 74.In some implementations, the completed midsole 14 can be coated with alayer of finishing material, including polyurethane, Barge® cement orthe like, or various corn or protein based materials, such as thecorn-based prolamine protein zein, to encapsulate the midsole 14 and/orto provide a desirable visual or tactile quality for the portions ofmidsole 14 that may include otherwise exposed resilin material. In thisrespect, the coating may also improve bonding by way of an adhesive orcement such that an additional primer is not needed. In general, asuitable coating or finishing material has comparable elastic propertiesto that of the resilin material over which it is applied, to preventseparation or cracking of the coating.

Turning to FIGS. 8-10, a variation of a lifestyle sneaker 110 is shownthat incorporates stylistic and assembly characteristics developedpreviously for use in more performance-oriented footwear. In one aspectthe upper used in connection with sneaker 110 can be a single piece or“seamless” upper or similar variations of the same. Such uppers can beof various woven or knit textile materials including various types of“technical” textile materials. Such textile materials can be of variousnatural or synthetic fibers and can be formed into the general shape ofupper 112 using various 3-D knitting processes. Alternatively, upper 112can be made of natural or synthetic leather, including in a non-seamlessconstruction similar to that which is shown above in FIGS. 1-3.

Sneaker 110 can include an outer of or including a resilin material of astructure similar to or resembling those incorporating a moldedelastomer foam. Such outers can include a midsole that is of a foammaterial, such as EVA or various composite materials including EVA orthe like, which can be injected into a mold in an uncured state andallowed to cure in the mold to achieve the desired shape, which caninclude more organic or amorphous forms than possible with a traditionalcut-and-buff midsole, such as that discussed above. Similarly, athermoplastic foam, such as EVA can be cut into a stock form andcompression molded under heat to achieve a similar effect. In someapplications, a molded midsole may comprise a number of different foammaterials, including in composition, density, etc., achieved bycompression molding different pieces of foam, cut to mutuallyinter-engage, in a single mold or by injecting different foams into amold simultaneously (e.g., dual-shot injection molding) or sequentially(insert molding). In a similar manner to that which is discussed abovein connection with FIG. 3, the present resilin material can be molded ofmultiple materials, in processes similar to either of theabove-described simultaneous or sequential molding processes to achievea molded resilin midsole 114 having multiple materials of differentcharacteristics for various purposes, including resilience, cushioning,wear-resistance, and the like, as discussed generally above and in theadditional examples below. In various implementations, the illustratedmidsole 114 can be of a foamed resilin material according to any of theabove-described variations thereof, which can be configured according tothe above description to achieve the desired properties of the material.

The illustrated molded resilin midsole 114 can include a contouredfootbed 176. Still further, the variations in the shape of midsole 114provided by molding can allow for adjustments in the shape of both theheel strike area 162, as well as the toe spring 168 to make midsole 114suitable for various purposes. In the present example, the midsole 114is illustrated as including both a heel wrap 178 and toe wrap 180defined by respective portions of the midsole 114 that are relativelythin and extend upwardly by between about ⅛″ and ½″ to provideadditional protection or traction along the portions of upper 112adjacent to heel 152 and fore-foot 156, such as along heel counter 136and toe tip 25. Additionally, the presence of heel wrap 178 and toe wrap180 can provide additional area for adhesion between midsole 114 andupper 112 in areas that are often subjected to relatively large amountsof stress.

As also shown in FIG. 10, midsole 114 can include various molded-infeatures along the outer portion thereof (i.e. the ground-facing sideopposite upper 112). Further, the outer of sneaker 110 may be configuredsuch that midsole 114 contacts the ground along portions thereof duringat least some use scenarios with no additional outsole 116 materialpresent in such areas. In this manner, midsole 114 can be configuredwith various forms of treads 184 or other traction-generating featuresin the areas of midsole 114 not covered by a portion of outsole 116. Inthe particular example of FIG. 10, midsole 114 is shown with noadditional outsole material thereon, such that the entire outer iscomprised of midsole 114, which includes treads 184 distributed alongthe entire ground-contacting portion thereof such that an outsole iseffectively defined on midsole 114. To achieve such a configuration in auseable manner, midsole 114 can be of a particular resilin material thatis optimized for abrasion resistance (while maintaining acceptablelevels of cushioning and/or rebound for the particular purpose for whichsneaker 110 is intended), at least along the outer portions thereofand/or along the portion defining treads 184 (which can be in thegeneral form shown or a variation thereof selected for variousperformance and stylistic considerations). In one non-limiting example,such a material may be achieved by adding fumed silica, or anotheraggregate or fiber having suitable properties, in an amount of up toabout 20% by weight to the purified resilin material prior tocross-linking. The addition of fumed silica to the resilin materialresults in a material, after cross-linking, that has a higher abrasionresistance and a higher durometer (as discussed below). In an example,the resulting material can be used for only a portion (e.g. theground-contacting portion or in an area that would otherwise comprise anoutsole) of midsole 114, with the remaining portion of midsole 114 beingformed of a more cushioning resilin material (i.e. without or with lessfumed silica). Such a structure can be achieved, in one example, byseparately forming the different portions of midsole 114 and bonding orcross-linking the materials together. In another example, a dual-shot orinsert molding construction can be employed, as discussed further below.In a still further example, midsole 114 can be of a “functionalgradient” construction in which a mold can be partially filled withpurified resilin, which can then be extensively cross-linked to derive afirst material. The mold can then be filled completely with additionalcross-linking to a lesser degree than that of the first material. Theresulting composite material could exhibit two different functionalproperties in the respective areas of different degrees ofcross-linking. Notably, this process can be carried out with or withoutthe above-mentioned aggregate material. In another example a solidresilin material of the same or different composition can be coated withan abrasion-resistant coating, including any of the above-describedcoatings.

In a similar manner to the implementation of midsole 14 discussed above,the midsole 114 illustrated in FIGS. 8-10 can incorporate a shank 170(FIG. 9) to provide rigidity and support for midsole 114 in the mannerdiscussed above. Midsole 114 can be adapted in one of a number of waysto securely receive shank 170 therein. In the illustrated example,midsole 114 can be molded over shank 170, which can be of a similarconstruction to the variations of shank 70 discussed above. In thismanner, the mold used to form midsole 114 can be adapted to retain shank170 therein prior to addition of the purified resilin protein prior tocross-linking, which can be achieved by including supports within moldthat can support shank 170 in a suspended position within the moldcavity. In various examples, the posts can be integrated with the moldand any holes preset from removal of the posts from the molded midsole114 can be filled with additional resilin material, including by theaddition of more purified and denatured resilin protein, which can becross-linked after filling of such holes. In another example, such holescan be filled with pre-fabricated plugs of resilin material, or theshank 170 can be supported on inserts prefabricated using a resilinmaterial or other compatible foam such that the posts become integratedwith midsole 114 during molding. In another example, shank 170 can beglued into a cavity in midsole 114 and can be covered by an insert of aresilin material sheet or other foam that can be glued over the shank170. In another example, shank 170 can be insert molded with midsole 114or adhered externally thereto along the outer surface thereof, in amanner similar to that which is described further below.

It will be appreciated that a midsole 114 of the present construction,including by formation of treads 184 along midsole 114 in place of aseparate outsole, may be suited for the present embodiment of sneaker inthe form of a lifestyle sneaker 110. In particular, the use of thepresently-described molded resilin material for the ground-contactingportion of sneaker 110 and, therefore, primary source of traction, mayresult in sneaker 110 having a level of grip that is well-suited foreveryday use, including walking and standing, while providing sufficientgrip for needed intermediate light running or jogging, the ultimateamount of grip provided by midsole 114 may not be sufficient for use inathletic activities carried out on smooth surfaces (such as basketball),where quick changes in lateral direction are needed (such as tennis), orin other activities where additional underfoot protection may be desired(such as trail running). The potentially-reduced grip of midsole 114 maybe offset, however, but gains in comfort by way of increased flexibilityand lightness that midsole 114 imparts on sneaker 110 overall. In asimilar manner, such gains in flexibility and lightness may also beadvantageous for certain athletic activities, including running andjogging, such that a variation of sneaker 110 with a midsole 114 similarto that of the present embodiment may be adapted for use as a runningsneaker. Such modifications may include the use of a resilin materialwith increased resilience or rebound, at least in the heel 152 area, orby the incorporation of small segments of outsole material inhigh-impact areas, such as in the heel strike 162 and toe spring 168areas. In one example, the outer portions of midsole 114 (e.g. thelowermost, ground-contacting portion and visible sidewalls) can be of asolid resilin material to provide the desired abrasion resistance andtraction, as well as to provide adequate support for any externalfeatures, while midsole 114 can have a core of a foamed resilinmaterial. To achieve such construction, midsole 114 can be formed inseparate interior and exterior portions that can be adhered orcrosslinked together into a single midsole 114.

As shown in FIGS. 11 and 12, a midsole 214,314 of the generalconfiguration discussed above can be adapted for use as an outer for asandal 210,319. In the example shown in FIG. 11, sandal 210 is in thegeneral form of a flip-flop, and midsole 214 is configured in a similarmanner to that which is shown in FIGS. 8-10, with the entire outer forsandal 210 comprising a molded resilin midsole 214, similar to midsole114, discussed above, including of the above-described foamed resilinmaterial. In this manner, midsole 214 can be made of a similar resilinmaterial to that of midsole 114 to provide desired abrasion resistance,with potential adjustments for other unique features or desiredcharacteristics for sandal 210. As shown, midsole 214 can be molded witha contoured footbed 276, including with a pronounced arch 264. Further,midsole 214 can be molded with holes 286 through which straps 288 canattach in a manner similar to other types of flip-flop style sandals.

In various implementations, holes 286 can be configured such that adisk- or T-shaped head of the straps 288 can be received within aportion thereof in a snap- or press-fit manner and/or to allow forpermanent fixation by way of adhesive or the like. As also discussedabove, midsole 214 can be molded with treads, similar to those shown inFIG. 10, in a pattern derived for the desired traction and stylisticconsiderations for sandal 210 such that no outsole is applied overmidsole 214 or such that only small portions of outsole material areapplied in specific areas of midsole 214, as also discussed above. Thedepicted straps 288 are exemplary only and can be configured differentlyfor purposes of style and fit. Straps 288 may be of any number ofmaterials or combinations thereof, including various plastics, includingthermoplastic elastomer, rubber, leather (or leather alternatives suchas mycelium material or the like), textile (both woven and variousnon-woven types), or molded or cut resilin material.

In an alternative embodiment, a flip-flop type sandal similar to thatwhich is shown in FIG. 11 can made from cut sheet stock of resilinmaterial in a laminated form similar to the midsole 14 used inconnection with sneaker 10, as described above. In such an embodiment,the ends of straps 288 can be embedded between layers with holes 286only extending partway through midsole 214.

A further embodiment of a sandal 310 is shown in FIG. 12, wherein sandal310 is in the form of a slide with a single strap-style upper 312affixed along the sidewalls 390 of midsole 314 from the medial side 358to the lateral side 360 thereof. As further shown, sandal 310 caninclude an outer comprising midsole 314 of a resilin material (includingof a foamed resilin material in whole or in part) and a separate outsole316 bonded with midsole 314 to define the ground-contacting surface forsandal 310. In a similar manner to that which is discussed above withrespect to FIG. 11, midsole 314 can be molded and can define a contouredfootbed 376, which can, as illustrated, exhibit additional contourcompared to footbed 276 in FIG. 11, although other configurations arepossible.

As also shown in FIG. 12, upper 312 can extend between respectiveportions of midsole 314 and outsole 316, which can provide additionalsurface area for attachment of upper 312 to midsole 314 and outsole 316,which may be achieved by way of adhesives or the like. In one example,upper 312 can extend entirely beneath midsole 314 to connect the medial358 and lateral 360 sides thereof, thereby further securing upper 312 tothe assembled outer. In this and similar constructions, midsole 314 canbe molded in such a way as to accommodate any portions of upper 312 thatextend therebeneath. The depicted upper 312 can take a number of knownalternative forms, consistent with the present disclosure, including byincorporating one or more straps with buckles or other adjustmentmechanisms and may include an additional strap or the like to extendaround the wearer's heel and/or a whole or partial enclosure over thewearer's toes (i.e., a clog). In any of these configurations, upper 312can be made from any number of materials or combinations thereof,including various plastics, including thermoplastic elastomer, rubber,leather (or leather alternatives such as mycelium material or the like),textile (both woven and various non-woven types), or molded or cutresilin material.

Turning to FIGS. 13-15, a further example of a sneaker 410 is shown thatincorporates a molded resilin midsole 414 generally similar to thatwhich is discussed above with respect to FIGS. 8-10 incorporated into anathletic sneaker. In such an embodiment, the upper 412 may be configuredto meet various performance-based criteria to make the sneaker 410 (andvariations thereof) suitable for different athletic activities orcombinations thereof according to various understood techniques and byincorporating various known features. Sneaker 410 according to thepresent embodiment can be so configured, such as by the generalreduction in seams to decrease weight and increase flexibility, as wellas by incorporating reinforcement 492 along the quarters 434 to helpincrease foot retention and stability. In the same manner, midsole 414can be configured to provide various performance characteristics desiredfor various types of athletic activity over the overall cushioning andcomfort that may be prioritized in implementations of midsole 14 and 114for the lifestyle sneakers 10 and 110 discussed above with respect toFIGS. 1-10.

In one example, sneaker 410 can be adapted for running by providing alightweight upper 412 with certain features promoting foot retention andallowing a tight fit without inducing discomfort. In such anarrangement, midsole 414 may be configured for reduced weight (includingby allowing reduction in the amount of midsole 414 covered by outer 416,as discussed above). Further, midsole 414 may be configured to provide adegree of cushioning sufficient to reduce fatigue, but may promotereturn of such energy rather than dissipation. Various compositions ofthe resilin material described herein may be used to provide such energyreturn, including by the particular selection of the resilin compositionand the cross-linking material and the non-aqueous solvent used for thesolvent exchange step discussed above. Still further, various materialscan be added to the resilin material prior to cross-linking to derive acomposite material.

In one example, such an additive can include fumed silica representingbetween about 2% to about 20% of the total material (including resilinprotein) used for midsole 414 by weight. In one embodiment, a resilinmidsole can incorporate fumed silica at about 10% by total weight. Thisand other aggregate additives can increase the overallstiffness/rigidity of midsole 414 and can, similarly, increase themodulus of elasticity of the midsole 414 material. In a further example,a midsole 414 comprised of different specific resilin materialcompositions in different areas thereof can further promote suitabilityfor various activities. In one example, a resilin material 414 a of arelatively higher density can be used in the heel 452 and mid-foot 454areas along the medial side 458, compared with a material 414 b throughthe remaining midsole, including the lateral side 460 to provide forstability control, particular in the management of over-pronation duringrunning. Other examples of such construction used in connection withprior foam midsoles can be similarly derived using various resilinmaterial compositions. Additionally, resilin material can be molded overvarious non-resilin inserts 494, including those of various elastomersto promote cushioning in various areas (such as above the heel strike462 and in the metatarsal head area 466 of the fore-foot 456). In somevariations, such inserts 494 can be in an internal pocket within midsole414 such that softer elastomers or even various liquids can be used foran insert 494 with appropriate protection thereof provided by midsole414. Various other types of athletic sneakers can similarly incorporatemidsoles of resilin material configured to provide characteristicsdesired by such activity, including for tennis, basketball, and thelike.

As discussed above, the resilin material used for midsole 414 can be afoamed resilin material or a laminated perforated structure, accordingto the various examples and configurations discussed above. In thismanner, the various portions of midsole 414 a and 414 b can be of foamedresilin materials having different properties (e.g. density) achieved byvariations in the foam, including cell size or the like. In a furtherexample, midsole 414 can be of a closed-cell foam structure that can beachieved, for example, by fabricating beads of resilin material (i.e.solid resilin material) according to various configurations for thedesired properties thereof. The resilin beads can be placed into a moldfor midsole 414 and re-crosslinked to join the beads together in theoverall shape for midsole 414. In this arrangement, the beads can be ofvarying sizes in a generally spherical shape such that, when the beadsare placed into the mold, voids are present between the beads with thebeads achieving sufficient mutual contact to enclose cells by way of thevoids. In this manner, beads of different sizes can be used together ina composite structure to provide the desired enclosed cellconfiguration. Further, different beads in different arrangements can beused in various areas of midsole 414 (including areas 414 a and 414 b,as shown in FIG. 14) to provide different properties for the closed cellresilin foam material.

As further shown in FIG. 15, midsole 414 can incorporate a moldedexternal shank 470 that extends along a portion of the outer surface ofmidsole 414 and is anchored to portions of the midsole 414 underlyingoutsole 416. Such implementations of shank 470 can be of an injectionmolded polymer (i.e., various plastics), carbon fiber reinforcedpolymer, or Kevlar reinforced polymer and can be of various shapes toachieve desired performance and stylistic characteristics. Shank 470, asshown, can be fabricated separate from midsole 414 and can be affixedtherewith using adhesives or the like with outsole 416 beingsubsequently assembled with midsole 414, including over shank 470. Inanother example, midsole 414 can be molded over shank 470 by positioningshank 470 in the midsole mold prior to the addition of the desiredresilin protein and crosslinking thereof. In various implementationsaccording to the illustrated midsole 414 and understood variationsthereof, midsole 114 may include various flex-notches 482 to providelocalized areas of increased flexibility, where desired.

Resilin material can be incorporated into additional types of footwearbased on applications of the above principles. As discussed above, shoeinsoles (e.g., insole 20) can be made of the present resilin material,including on the uppermost surface (due to the potentialbiocompatibility of resilin materials). Such insoles can incorporatereinforcement, including by molding resilin material over an insert orscrim to increase the strength of the insole and its resistance againsttearing and the like. Insoles of variations of the present resilinmaterials can be utilized in practically any type of footwear, includingdress shoes, work shoes, boots, etc. Still further, inserts of moldedresilin material can be incorporated into various types of midsolesresembling or otherwise similar to existing types of midsoles. In onesuch example, a molded insert of resilin material optimized forcushioning can be incorporated into the interior of a dress shoemidsole, such as by the fabrication of a generally traditional midsoleof leather or the like with an internal cavity for receiving the resilininsert therein in a concealed manner. Similar inserts can be similarlyincorporated into other types of midsoles, including athletic midsolesof a foam material. Still further, a molded resilin material can be usedas an outer for a dress shoe in a similar manner to the use of midsole114 as the outer for the lifestyle sneaker 110, discussed above.Additionally, resilin materials of the types discussed herein can beused to replace various foams used elsewhere in various types of shoes,including within the tongues 30 and the collar 38 areas thereof or otherareas where padding is incorporated therein.

As mentioned above, the shape and configuration of the above-describedportions of the upper are exemplary only and can be altered to achievedifferent appearances, as well as different fit and performancecharacteristics (flexibility, support, weight, etc.).

In some respects, the properties of the resilin that are generallycomparable to elastomers can allow the above assembly to be completedusing the above techniques with parameters and equipment identical to orcomparable to those used in assembly of sneaker midsoles of elastomer,resulting in a similar appearance and the efficiencies of usingestablished techniques and existing machinery. In this respect, theresilin material is generally not thermoplastic such that molding iscarried out in a different manner than with some typical elastomers. Theresilin material, however, may be amenable to other processing andfabrication techniques used for elastomer that may be useful infabricating the footwear disclosed herein.

It is to be appreciated that the above techniques and fabricationmethods using the resilin material can also be used to fabricate othertypes of footwear, including the various types (slippers, sandals,moccasins, boat shoes) mentioned above by using techniques generallysimilar to those used to make portions of such footwear from elastomers,while taking advantage of the numerous additional properties of theresilin material to provide additional benefits for such footwear andthe construction thereof according to the principles and variationsdescribed above. In this manner, the midsoles of various styles of dressshoes, boots, and the like can also be made of the present resilinmaterial. In one application, resilin material may be derived andprocessed to resemble a “crepe sole” that is used in certain styles ofboots (e.g., desert boots) and dress-shoes. Other similar applicationsare also possible.

It will be understood by one having ordinary skill in the art thatconstruction of the described device and other components is not limitedto any specific material. Other exemplary embodiments of the devicedisclosed herein may be formed from a wide variety of materials, unlessdescribed otherwise herein.

It is also important to note that the construction and arrangement ofthe elements of the articles, as shown, in the examples above areillustrative only. Although only a few embodiments of the presentinnovations have been described in detail in this disclosure, thoseskilled in the art who review this disclosure will readily appreciatethat many modifications are possible (e.g., variations in sizes,dimensions, structures, shapes and proportions of the various elements,values of parameters, mounting arrangements, use of materials, colors,orientations, etc.) without materially departing from the novelteachings and advantages of the subject matter recited. For example,elements shown as integrally formed may be constructed of multiple partsor elements shown as multiple parts may be integrally formed, theoperation of the interfaces may be reversed or otherwise varied, thelength or width of the structures and/or members or connector or otherelements of the system may be varied, the nature or number of adjustmentpositions provided between the elements may be varied. Accordingly, allsuch modifications are intended to be included within the scope of thepresent innovations. Other substitutions, modifications, changes, andomissions may be made in the design, operating conditions, andarrangement of the desired and other exemplary embodiments withoutdeparting from the spirit of the present innovations.

It will be understood that any described processes or steps withindescribed processes may be combined with other disclosed processes orsteps to form structures within the scope of the present device. Theexemplary structures and processes disclosed herein are for illustrativepurposes and are not to be construed as limiting.

It is also to be understood that variations and modifications can bemade on the aforementioned structures and methods without departing fromthe concepts of the present device, and further it is to be understoodthat such concepts are intended to be covered by the following claimsunless these claims by their language expressly state otherwise.

The above description is considered that of the illustrated embodimentsonly. Modifications of the device will occur to those skilled in the artand to those who make or use the device. Therefore, it is understoodthat the examples shown in the drawings and described above are merelyfor illustrative purposes and not intended to limit the scope of thearticle, which is defined by the following claims as interpretedaccording to the principles of patent law, including the Doctrine ofEquivalents.

What is claimed:
 1. A method for making an article of footwear,comprising: placing a purified recombinant resilin composition in a moldwith a cross-linking solution; incubating the recombinant resilincomposition in the cross-linking solution to generate a solid resilinmaterial; fabricating a midsole including at least a portion of thesolid resilin material; and assembling the midsole with an upper.
 2. Themethod for making the article of footwear of claim 1, furthercomprising: prior to fabricating the midsole, subjecting the solidresilin material to a solvent exchange process to substantially removethe cross-linking solution and configure the solid resilin material as asolid resilin material comprising a cross-linked recombinant resilin anda polar nonaqueous solvent.
 3. The method for making an article offootwear of claim 2, wherein the polar nonaqueous solvent is glycerol.4. The method for making an article of footwear of claim 1, wherein: themold defines a shape of the midsole; and fabricating the midsoleincludes removing the solid resilin material from the mold such that theentire midsole is of the solid resilin material.
 5. The method formaking an article of footwear of claim 4, wherein the shape of themidsole defined by the mold is configured to compensate for subsequentshrinking of the solid resilin material by between about 20% and about40%.
 6. The method for making an article of footwear of claim 4,wherein: the shape of the midsole defined by the mold includes at leastone integral ground-contacting feature; and the method does not includeaffixing an outsole material over the ground-contacting feature.
 7. Themethod for making an article of footwear of claim 1, wherein theincubation is carried out at a temperature of at least 60° C. for atleast 15 minutes, at least 30 minutes, at least 45 minutes, at least 60minutes, at least 90 minutes, or at least 2 hours.
 8. The method formaking an article of footwear of claim 1, wherein the purifiedrecombinant resilin material is a solution that further includes afoaming agent.
 9. The method for making an article of footwear of claim8, wherein the foaming agent includes a mixture of ammonium persulfateand xanthan gum.
 10. The method for making an article of footwear ofclaim 8, wherein the purified recombinant resilin solution furtherincludes fumed silica and sodium bicarbonate.
 11. The method for makingan article of footwear of claim 8, wherein the purified recombinantresilin solution is vortexed and heated to promote development of airbubbles therein prior to being placed in the mold.
 12. The method formaking an article of footwear of claim 11, wherein incubating therecombinant resilin composition in the cross-linking solution togenerate a solid resilin material causes the air bubbles to form opencells within a matrix of the solid resilin material.
 13. The method formaking an article of footwear of claim 1, further including fabricatingan insole from a remaining portion of the solid resilin material. 14.The method for making an article of footwear of claim 13, furtherincluding placing the insole within an interior of the upper and over aportion of the midsole.
 15. A method for making an article of footwear,comprising: placing a purified recombinant resilin composition in a moldwith a cross-linking solution; incubating the recombinant resilincomposition in the cross-linking solution to generate a solid resilinmaterial; fabricating an insole including at least a portion of thesolid resilin material; and assembling the insole within an upper andover a portion of a midsole of the article of footwear.
 16. The methodfor making the article of footwear of claim 15, further comprising:prior to fabricating the insole, subjecting the solid resilin materialto a solvent exchange process to substantially remove the cross-linkingsolution and configure the solid resilin material as a solid resilinmaterial comprising a cross-linked recombinant resilin and a polarnonaqueous solvent.
 17. The method for making an article of footwear ofclaim 15, wherein the purified recombinant resilin material is asolution that further includes a foaming agent.
 18. The method formaking an article of footwear of claim 15, wherein fabricating theinsole further includes applying a material layer over the portion ofthe solid resilin material.
 19. The method for making an article offootwear of claim 15, wherein fabricating the insole includes cutting aportion of a sheet of the solid resilin material into a desired shape ofthe insole.
 20. A method for making an article of footwear, comprising:placing a purified recombinant resilin composition in a mold with across-linking solution; incubating the recombinant resilin compositionin the cross-linking solution to generate a solid resilin material;fabricating a cushioning element of the article of footwear including atleast a portion of the solid resilin material; and assembling thecushioning element insole with at least an upper of the article offootwear.